Three bioreactor system for the cultivation of mammalian cells

ABSTRACT

The present invention relates to large-scale bioreactors having at least two impellers, large-scale bioreactor systems and methods for the large scale cultivation and propagation of mammalian cells using these bioreactors.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/148,503, filed Aug. 23, 2011, which is anational stage application of International Application No.PCT/EP2010/000783, filed Feb. 9, 2010, which claims priority to EuropeanApplication No. 09001755.9, filed Feb. 9, 2009, each of which isincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to bioreactors and methods for the largescale cultivation of mammalian cells using these bioreactors.

BACKGROUND OF THE INVENTION

It is important in mammalian cell culture processes to maintain thephysicochemical environment in view of dissolved oxygen, culture pH,temperature and shear sensitivity. Also the maintenance of thenutritional environment is important. The maintenance of the cultivationconditions limits the possibility to perform large scale culturing ofmammalian cells. Especially concentration gradients can inhibit the cellgrowth of mammalian cells in large-scale bioreactors.

One of the objects of the present invention is to provide bioreactorsand methods, which allow the cultivation of mammalian cells in largescale volumes. Furthermore, it is an object of the present invention toprovide bioreactors and methods, which allow the cultivation ofmammalian cells under optimal conditions, even if grown in large scalevolumes and therefore allow a process performance and product qualityindependent of the size of the bioreactor.

It is an object of the present invention to provide large-scalebioreactors which allow the cultivation of mammalian cells in ahomogenous environment with respect to process parameters such as pH,dissolved oxygen tension (DOT) and temperature, maintaining a well mixedcell suspension and blending nutrient feeds within the bioreactor.

Furthermore it is an object of the present invention to provide devicesand methods which allow the production of mammalian cells and productsof the mammalian cells, especially proteins, peptides, antibiotics oramino acids, synthesised by the mammalian cells, in a large-scalemanner.

The present invention solves the technical problems underlying thepresent invention by the provision of bioreactors, bioreactor systemsand methods for the cultivation of eukaryotic cells, especially ofmammalian cells, according to the claims.

The present invention solves the technical problem underlying thepresent invention especially by the provision of a bioreactor for thecultivation of mammalian cells, characterised in that said bioreactorhas at least two impellers. Furthermore, the present invention solvesthe technical problem underlying the present invention by the provisionof a method for the cultivation and propagation of mammalian cellscharacterised in that at least one mammalian cell is cultivated undersuitable conditions and in a suitable culture medium in a bioreactor,which has at least two impellers. Furthermore, the present inventionsolves the technical problems underlying the present invention by theprovision of a bioreactor system for the cultivation of mammalian cellscharacterised in that a) a first bioreactor with a volume of at least500 l is connected with b) a second bioreactor with a volume of at least2000 l, which has a volume greater than the first bioreactor and whereinthe second bioreactor with a volume of at least 2000 l is connected withc) a third bioreactor having at least two impellers and a volume of atleast 10 000 l, which has a volume greater than the second bioreactor.

The present invention solves the technical problem underlying thepresent invention furthermore by the provision of a method to cultivateand propagate mammalian cells, characterised in that a) at least onemammalian cell is cultivated under suitable conditions and in a suitableculture medium in a first bioreactor with a volume of at least 500 l, b)the medium containing the cells obtained by propagation of the at leastone mammalian cell is transferred into a second bioreactor with a volumeof at least 2000 l, c) the transferred cells are cultivated in thesecond bioreactor with a volume of at least 2000 I, d) the mediumcontaining the cells obtained in step c) is transferred into a thirdbioreactor with a volume of at least 10 000 l and e) the transferredcells are cultivated in the third bioreactor with a volume of at least10 000 l.

According to the invention, the cultivated cells are eukaryotic cells,preferably animal cells, more preferably mammalian cells. The mammaliancells can be for example human cell lines, mouse myeloma (NS0)-celllines, Chinese hamster ovary (CHO)-cell lines or hybridoma-cell lines.Preferably the mammalian cells are CHO-cell lines.

Preferably the cultivated cells are used to produce antibodies, morepreferably monoclonal antibodies, and/or recombinant proteins, morepreferably recombinant proteins for therapeutic use. Of course the cellsmay produce peptides, amino acids, fatty acids or other usefulbiochemical intermediates or metabolites. According to the invention thetarget concentration of the proteins produced by the cultivated cells ismore than 0.5 g/l, preferably more than 2.0 g/l and most preferred morethan 10.0 g/l. The method according to the invention can be used as abatch or in a fed-batch process. Although the cell-culture-medium usedin the method according to the invention is preferably protein freemedium, the design does not exclude the use of protein containingstreams.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention a bioreactor is a biocompatible tank orvessel having additional equipment, for example impellers, baffles,spargers and/or ports, which specifically allows for the cultivation andpropagation of mammalian cells. Preferably the tank or vessel is in theform of a tube, having on both ends of the tube, which build preferablythe top and the bottom of the tank, plates. The plates are called headplate and base plate. In a particularly preferred embodiment of thepresent invention the base plate is an American Society of MechanicalEngineers Flanged and Dished (ASME F&D) designed base plate. Thehead-plate design preferably accommodates a manway or is preferably aflanged head plate to allow access/removal of the impellers.

The total tank height is the tangential line from the inner tank side ofthe base to the inner tank side of the head of the tank.

The freeboard height is defined as the length of straight side above theliquid head when the bioreactor is filled to it's operating volume. Aminimum freeboard height is necessary taking into account the extent offoam build up during operation, gas hold up at maximum allowed agitationand aeration and errors in metering liquid.

The bioreactor according to the invention has a volume of preferably atleast 500 l, more preferably of at least 1000 l, more preferably of atleast 4000 l, even more preferably of at least 10 000 l, even morepreferably of at least 20 000 l. Most preferably the bioreactoraccording to the invention has a volume of 1000 l, 1307 l, 4000 l, 5398l, 20 000 l or 27 934 l.

Preferably, the bioreactor has a maximum volume of 100 000 l, morepreferably the bioreactor has a maximum volume of 50 000 l, mostpreferably the bioreactor has a maximum volume of 30 000 l.

The design of the bioreactors according to the present invention ensuresa homogenous environment with respect to process parameters such as pH,dissolved oxygen tension (DOT) and temperature, maintaining a well mixedcell suspension and blending nutrient feeds within the bioreactor. Thisprovides the necessary physicochemical environment for optimal cellgrowth, product accumulation and product quality. The design of thebioreactors according to the present invention furthermore ensures themaintenance of geometric similarity. This allows a scale down model tobe developed at 12 liter laboratory and 500 liter pilot scales.

The bioreactor for the cultivation of mammalian cells according to theinvention has at least two impellers. More preferably, the bioreactorhas two impellers, even more preferably a top impeller and a bottomimpeller.

The bioreactor for the cultivation of mammalian cells according to theinvention has preferably at least one top impeller and at least onebottom impeller, wherein the top impeller is preferably a hydrofoilimpeller.

The bioreactor for the cultivation of mammalian cells according to theinvention has preferably at least one top impeller and at least onebottom impeller, wherein the top impeller is a hydrofoil impeller.

The bioreactor for the cultivation of mammalian cells according to theinvention has preferably a volume of at least 1000 l and at least onetop impeller and at least one bottom impeller, wherein the top impelleris a hydrofoil impeller.

The bioreactor for the cultivation of mammalian cells according to theinvention has preferably a volume of at least 4000 l and at least onetop impeller and at least one bottom impeller, wherein the top impelleris preferably a hydrofoil impeller.

The bioreactor for the cultivation of mammalian cells according to theinvention has preferably a volume of at least 4000 l and at least onetop impeller and at least one bottom impeller, wherein the top impelleris a hydrofoil impeller.

In a preferred embodiment of the invention the top impeller is ahydrofoil impeller. The top impeller can be used preferably to providestrong bulk mixing.

In a preferred embodiment of the invention the bottom impeller is ahydrofoil impeller. In a preferred embodiment of the invention the topimpeller and the bottom impeller are a hydrofoil impeller.

In a preferred embodiment of the invention at least the top impeller isa hydrofoil impeller. In a preferred embodiment of the invention allimpellers are hydrofoil impellers.

According to a preferred embodiment of the invention the bottom impelleris a high-solidity pitch-blade impeller or a high-solidity hydrofoilimpeller. The bottom impeller can be used preferably for the dissipationof sparged gas.

Preferably, the hydrofoil impellers provide much greater liquid motion,resulting in a greater bulk-mixing, for a given amount of power input.This can also depend of the flow number (N_(q)).

Preferably, non-hydrofoil impellers can provide liquid motion but atgreater power inputs. This can have consequences on the health ofshear-sensitive mammalian cells.

In a preferred embodiment of the invention the hydrofoil impeller is adown-flowing impeller or a up-flowing impeller.

In a preferred embodiment of the invention the top impeller is adown-flowing impeller. In a preferred embodiment of the invention thetop impeller is a down-flowing axial hydrofoil impeller.

In a preferred embodiment of the invention the pulling downcharacteristics of the top impeller are used to mix the well aeratedliquid surface with the liquid bulk.

In a preferred embodiment of the invention the hydrofoil impeller is ahigh efficiency hydrofoil impeller. In a preferred embodiment of theinvention the hydrofoil impeller is a Chemineer—model SC-3 impeller, aLIGHTNIN—model A310 or A510 impeller, a Promix—model PHF series impelleror a Cleaveland Eastern Mixers impeller.

In a preferred embodiment of the invention the top impeller is a highefficiency hydrofoil impeller. In a preferred embodiment of theinvention the top impeller is a Chemineer—model SC-3 impeller, aLIGHTNIN—model A310 or A510 impeller, a Promix—model PHF series impelleror a Cleaveland Eastern Mixers impeller.

The top impeller is preferably a three-bladed hydrofoil design impeller,for example a A310-type impeller from LIGHTNIN. The bottom impeller ispreferably a four-pitched-bladed high-solidity impeller, for example ofthe A315-type from LIGHTNIN. The impeller to tank diameter ratio of thetop impeller (D_(top)/T) and/or of the bottom impeller (D_(bottom)/T) ispreferably at least 0.35 and at most 0.55, more preferably at least 0.40and at most 0.48, and most preferably at least 0.44, and at most 0.46. Adiameter greater than 0.5 results in disruption in axial flow, hencepoor agitation and aeration.

The top impeller power number (N_(p)) is preferably at least 0.1 and atmost 0.9, more preferably at least 0.25 and at most 0.35, mostpreferably 0.3. The top impeller flow number (N_(q)) is preferably atleast 0.4 and at most 0.9, more preferably at least 0.50 and at most0.60, most preferably 0.56. The bottom impeller power number (N_(p)) ispreferably at least 0.5 and at most 0.9, more preferably at least 0.70and at most 0.80, most preferably 0.75. The bottom impeller flow number(N_(q)) is preferably at least 0.50 and at most 0.85, more preferably atleast 0.70 and at most 0.80, most preferably 0.73.

The impeller power number (N_(p)) is a measure of an impeller efficiencyto impart the kinetic energy of the rotating impeller blades to thefluid. It is important in quantifying the gas dispersion. The impellerflow number (N_(q)) is a measure of pumping ability of the impeller andis important in quantifying fluid bulk movement.

The agitation rate of the at least two impellers is dependent on thescale. However, in a particularly preferred embodiment of the inventionthe agitation rate of the at least two impellers is at most 200 roundsper minute (rpm), more preferably at most 165 rpm.

The impeller spacing (D_(s)) is the space between the at least twoimpellers. It is in a particularly preferred embodiment of the inventionat least 1×the diameter of the bottom impeller (D_(bottom)) and at most2×D_(bottom), more preferably it is 1.229×D_(bottom) or 2×D_(bottom).This will allow both impellers to remain submerged at the lowestpost-inoculation volume.

The liquid height above the upper impeller (D_(o)) is in a particularlypreferred embodiment of the invention at least 0.3×the diameter of thetop impeller (D_(top)) and at most 2.5×D_(top). More preferably it is atleast 0.5×D_(top) and at most 2.0×D_(top).

The bottom clearance (D_(c)) is the clearance between the tank bottomand the centre-line of the bottom impeller. In a particularly preferredembodiment of the invention it is at least 0.35×D_(bottom), morepreferably it is either 0.4×D_(bottom) or 0.75×D_(bottom).

The design of the impellers in the bioreactor according to the presentinvention provides optimal hydrodynamic characteristics in terms of bulkmixing, gas dispersion and low shear. The mammalian cells are kept in ahomogeneous suspension by agitation via the impeller system according tothe present invention.

The design of the impellers in the bioreactor according to the presentinvention provides rapid mixing, maintain homogeneity, maintainmammalian cells in suspension and gas bubble dispersion. The design ofthe impellers in the bioreactor according to the present inventionminimises cell damage through shear forces originating from impellergeometry and eddies or vortices created behind the impeller blades.

In a particularly preferred embodiment of the present invention, the atleast two impellers are a top driven agitator system.

The supply of air, especially compressed air, or specific gases,preferably oxygen, nitrogen and/or CO₂ is realised preferably through atleast one sparger.

The bioreactor according to the invention has preferably at least onesparger, more preferably the bioreactor has one sparger or two spargers.The bioreactor according to the invention has preferably two spargers.Preferably the bioreactor has at least one sparger with a pipe-geometry.Preferably the at least one sparger is of the flute-type or is asintered sparger. Preferably the at least one sparger is of theflute-type. In particularly preferred embodiment of the presentinvention a crescent pipe is explored. The curvature of the crescent ispreferably 0.8×D_(bottom). In order to aid installation and removal fromside ports of the bioreactor the crescent circumference is preferably240° of the complete circumference of 0.8×D_(bottom) ring.

The at least one sparger provides sufficient oxygen mass transfer(characterised by K_(L)a) to meet the oxygen demand of the culture. Theat least one sparger provides a K_(L)a up to 20 h⁻¹ for culturesreaching up to 20×10⁶ cells per ml with an oxygen uptake rate of 5mmol/l per hour. Two spargers used as a dual sparger system allow theremoval of dissolved CO₂ and control of dissolved oxygen tension (DOT).Fluted spargers offer the benefits of easier cleaning in place (CIP) andsterilisation in place (SIP), aids with dCO₂ stripping and reducedoperational costs as it is multiple use. Sintered spargers providehigher K_(L)a values. The lower intrinsic K_(L)a value with the flutedsparge design can be compensated by the use of oxygen enriched air. Thegas flow rates are scaled up on the basis of constant superficial gasvelocity.

It is important in large scale cultivation of mammalian cells tomaintain a homogenous physicochemical environment in terms of dissolvedoxygen, culture pH, and temperature, and dissolved CO₂, nutrient andmetabolite concentration gradients. Whilst ensuring the physicochemicalenvironment is homogenous through using appropriate agitation andaeration, it is important to ensure the selected operating agitation andaeration conditions do not produce adverse shear environment. Theappropriate balance between ensuring homogenous environment that willpromote good cell growth and productivity of mammalian cell cultureprocesses whilst minimising the adverse effects of shear environment isdealt with in this invention. This is achieved through prescribingspecific bioreactor geometries, impeller design and positioning, spargerdesign and positioning and specific operating limits for agitation andaeration rates.

The major damage to mammalian cells in stirred and sparged bioreactorscomes from interfacial shear. Interfacial shear occurs as sparged gasbubbles coalesce and burst [ref: Ma N, Koelling K W, Chalmers J J.Biotechnol Bioeng. 2002 Nov. 20; 80(4):428-37. Erratum in: BiotechnolBioeng. 2003 Feb. 5; 81(3):379]. Thus minimising sparged gas flows andexcessive build up of foam is desirable. The interfacial shear can beminimised through a combination of approaches first by promoting surfaceaeration through good mixing of the liquid surface with the liquid bulkand secondly higher oxygen driving force by segregated oxygenation ofcultures through the preferably two spargers.

The prescribed positioning of the hydrofoil impeller, particularly theliquid height above the upper impeller, D_(o) preferably being around0.5×D_(top), below the liquid surface can aid strong and continuousexchange of the liquid surface with the liquid bulk thereby mixing thewell oxygenated liquid surface with less oxygenated liquid bulk. Theprescribed impeller spacing, preferably being D_(s)=1×D_(bottom) to2×D_(bottom) can permit the down-flow of liquid generated by the upperimpeller to feed fluid flow into the lower impeller thereby ensuring thewhole fluid bulk is well-mixed and separate mixing zones are not made.The prescribed impeller bottom clearance, preferably being D_(c)=0.35×Dto 0.75×D can ensure that the bulk flow is able to deflect off thecurved ASME F&D base and rise upwards along the walls of the bioreactor.

The segregation of the ‘on-demand’ oxygenated sparged gas through thecontrol sparger from the non-oxygenated sparged gases (such as CO₂, airand nitrogen ballasts) through a ballast sparger can allow greaterresidence time and path length of highly oxygenated sparge gas bubblesin the fluid bulk before disengaging out of the fluid bulk and into theheadspace. This can permit greater oxygen transfer rates to be providedfor a given volumetric mass transfer coefficient, k_(La). The residencetime and path length of the sparged gas bubbles can be extended furtherthrough specifying down-flowing axial hydrofoil impellers thatcontinuingly pull the liquid surface and liquid bulk down.

The bioreactor according to the invention has preferably at least onebaffle, more preferably at least two baffles. The bioreactor accordingto the invention has most preferably four baffles.

Baffles are vertical radially located plates. Baffles are used toprevent the formation of a funnel or vortex formation.

In a preferred embodiment of the invention, the length of the at leastone baffle is 1.1×the total straight height (H) of the bioreactor. Thewidth of the baffle (W) is preferably 0.1×the internal diameter of thetank (T). The baffle clearance (W_(c)) is preferably 0.01×the internaldiameter of the tank (T). The height of at least one baffle (H_(baffle))is preferably 1.1×the total straight height (H)—the height of thebioreactor-head (H_(h)). Therefore H_(baffle) is preferably calculatedaccording to the formula H_(baffle)=1.1×H−H_(h).

The thickness of the at least one baffle is not specified but thethickness needs to ensure rigidity to the radial component of the fluidflow. Additionally thickness needs to ensure the baffle plates are notwarped during SIP thereby affecting the baffle to tank wall clearance.

The bioreactor according to the invention has preferably at least twoports for alkali addition. More preferably, the bioreactor has two portsfor alkali addition. Most preferably, the bioreactor has two ports foralkali addition, wherein the first port is located at the central lineof the bottom impeller and the second port is located at the centralline of the top impeller. Preferably the pH probes are locateddiametrically opposite the alkali addition points into the bioreactor.

In a preferred embodiment of the invention, the bioreactor has a volumeof 1000 l. The head volume (V_(h)) of a 1000 l bioreactor is preferablyat least 45 l and at most 65 l, more preferably the head volume is 55 l.The base volume (V_(b)) of the 1000 l bioreactor is preferably at least45 l and at most 65 l, more preferably the base volume is 55 l. The tankinternal diameter (T) of the 1000 l bioreactor according to theinvention is preferably at least 850 mm and at most 900 mm, morepreferably the tank internal diameter is 864 mm. The tankcross-sectional area (A) of the 1000 l bioreactor according to theinvention is preferably at least 0.55 m² and at most 0.65 m², morepreferably the tank cross-sectional area is 0.586 m². The head height(H_(h)), which is the height of the head-plate, and/or the base height(H_(b)), which is the height of the base-plate, of the bioreactor with avolume of 1000 l according to the invention is preferably at least 120mm and at most 180 mm, more preferably the head height and/or the baseheight is 151 mm. The total tank height of the 1000 l bioreactoraccording to the invention is preferably at least 2000 mm and at most2600 mm, more preferably the total tank height is 2347 mm. The topimpeller diameter (D_(10p)) and/or the bottom impeller diameter(D_(bottom)) of the 1000 l bioreactor according to the invention ispreferably at least 350 mm and at most 400 mm, more preferably the topimpeller diameter and/or the bottom impeller diameter is 381 mm. Theclearance between the tank bottom and centre-line of the bottom impeller(D_(c)) is for the 1000 l bioreactor according to the invention,preferably at least 120 mm and at most 180 mm, more preferably theclearance is 152 mm. The distance between the at least two impellers,also known as impeller separation (D_(s)) is for the 1000 l bioreactor,according to the invention, preferably at least 730 and at most 790 mm,more preferably the impeller separation is 762 mm. The impeller shaftdiameter for the 1000 l bioreactor according to the invention ispreferably at least 102 mm and at most 152 mm. If the 1000 l bioreactoraccording to the invention has baffles, the length of the baffles ispreferably at least 2000 mm and at most 2400 mm, more preferably thelength is 2250 mm. The width of the baffles for the 1000 l bioreactoraccording to the invention is preferably at least 70 mm and at most 100mm, more preferably the width is 86 mm. The baffle clearance for the1000 l bioreactor according to the invention is preferably at least 7 mmand at most 11 mm, more preferably the baffle clearance is 9 mm. Thebaffle height (H_(baffle)) for the 1000 l bioreactor according to theinvention is preferably at least 2000 mm and at most 2200 mm, morepreferably the baffle height is 2099 mm. The 1000 l bioreactor accordingto the invention has preferably at least one sparger, more preferably ithas one sparger. The at least one sparger of the 1000 l bioreactoraccording to the invention has preferably an orifice- or pore-size of atleast 1.5 mm and at most 2.5 mm, more preferably the orifice- orpore-size is 2 mm. The orifice- or pore-number is preferably at least 20and at most 40, more preferably the orifice- or pore-number is 30. Thesparger length (S_(L) is preferably at least 150 mm and at most 550 mm,more preferably the sparger length is 305 mm. The sparger to tank bottomclearance (S_(c)) of the 1000 l bioreactor according to the invention ispreferably at least 50 mm and at most 75 mm, more preferably the spargerto tank bottom clearance is 64 mm. The sparger to bottom impellerclearance (D_(c)−S_(c)) of the 1000 l bioreactor according to theinvention is preferably at least 75 mm and at most 100 mm, morepreferably the sparger to bottom impeller clearance is 88 mm.

In a preferred embodiment of the invention, the bioreactor has a volumeof 4000 l. The head volume (V_(h)) of a 4000 l bioreactor is preferablyat least 340 l and at most 370 l, more preferably the head volume is 359l. The base volume (V_(h)) of the 4000 l bioreactor is preferably atleast 340 l and at most 370 l, more preferably the base volume is 359 l.The tank internal diameter (T) of the 4000 l bioreactor according to theinvention is preferably at least 1600 mm and at most 1650 mm, morepreferably the tank internal diameter is 1626 mm. The tankcross-sectional area (A) of the 4000 l bioreactor according to theinvention is preferably at least 1.90 m² and at most 2.30 m², morepreferably the tank cross-sectional area is 2.076 m². The head height(H_(h)) and/or the base height (H_(b)) of the bioreactor with a volumeof 4000 l according to the invention is preferably at least 260 mm andat most 300 mm, more preferably the head height and/or the base heightis 282 mm. The total tank height of the 4000 l bioreactor according tothe invention is preferably at least 2300 mm and at most 3100 mm, morepreferably the total tank height is 2817 mm. The top impeller diameter(D_(top)) and/or the bottom impeller diameter (D_(bottom)) of the 4000 lbioreactor according to the invention is preferably at least 680 mm andat most 740 mm, more preferably the top impeller diameter and/or thebottom impeller diameter is 710 mm. The clearance between the tankbottom and centre line of the bottom impeller (D_(c)) is for the 4000 lbioreactor according to the invention, preferably at least 500 mm and atmost 560 mm, more preferably the clearance is 531 mm. The distancebetween the at least two impellers, also known as impeller separation(D_(s)) is for the 4000 l bioreactor, according to the invention,preferably at least 840 mm and at most 900 mm, more preferably theimpeller separation is 872 mm. The impeller shaft diameter for the 4000l bioreactor according to the invention is preferably at least 51 mm andat most 64 mm. If the 4000 l bioreactor according to the invention hasbaffles, the length of the baffles is preferably at least 2200 mm and atmost 2600 mm, more preferably the length is 2477 mm. The width of thebaffles for the 4000 l bioreactor according to the invention ispreferably at least 150 mm and at most 180 mm, more preferably the widthis 163 mm. The baffle clearance is for the 4000 l bioreactor accordingto the invention preferably at least 12 mm and at most 20 mm, morepreferably the baffle clearance is 16 mm. The baffle height (H_(baffle))for the 4000 l bioreactor according to the invention is preferably atleast 2100 mm and at most 2300 mm, more preferably the baffle height is2195 mm. The 4000 l bioreactor according to the invention has preferablyat least one sparger, more preferably it has one sparger. The at leastone sparger of the 4000 l bioreactor according to the invention haspreferably an orifice- or pore-size of at least 1.5 mm and at most 2.5mm, more preferably the orifice- or pore-size is 2 mm. The orifice- orpore-number for the 4000 l bioreactor according to the invention ispreferably at least 80 and at most 120, more preferably the orifice- orpore-number is 100. The sparger length (S_(L)) is preferably at least250 mm and at most 800 mm, more preferably the sparger length is 568 mm.The sparger to tank bottom clearance (S_(c)) of the 4000 l bioreactoraccording to the invention is preferably at least 315 mm and at most 360mm, more preferably the sparger to tank bottom clearance is 337 mm. Thesparger to bottom impeller clearance (D_(c)−S_(c)) of the 1000 lbioreactor according to the invention is preferably at least 180 mm andat most 205 mm, more preferably the sparger to bottom impeller clearanceis 194 mm.

In a preferred embodiment of the invention, the bioreactor has a volumeof 20 000 l. The head volume (V_(h)) of a 20 000 l bioreactor ispreferably at least 1600 l and at most 2000 l, more preferably the headvolume is 1803 l. The base volume (V_(b)) of the 20 000 l bioreactor ispreferably at least 1600 l and at most 2000 l, more preferably the basevolume is 1803 l. The tank internal diameter (T) of the 20 000 lbioreactor according to the invention is preferably at least 2500 mm andat most 3000 mm, more preferably the tank internal diameter is 2794 mm.The tank cross-sectional area (A) of the 20 000 l bioreactor accordingto the invention is preferably at least 5.8 m² and at most 6.5 m², morepreferably the tank cross-sectional area is 6.131 m². The head height(H_(h)) and/or the base height (H_(b)) of the bioreactor with a volumeof 20 000 l according to the invention is preferably at least 460 mm andat most 500 mm, more preferably the head height and/or the base heightis 485 mm. The total tank height of the 20 000 l bioreactor according tothe invention is preferably at least 4800 mm and at most 5100 mm, morepreferably the total tank height is 4933 mm. The top impeller diameter(D_(top)) and/or the bottom impeller diameter (D_(bottom)) of the 20 000l bioreactor according to the invention is preferably at least 1100 mmand at most 1300 mm, more preferably the top impeller diameter and/orthe bottom impeller diameter is 1219 mm. The clearance between the tankbottom and centre line of the bottom impeller (D_(c)) is for the 20 000l bioreactor according to the invention, preferably at least 890 mm andat most 945 mm, more preferably the clearance is 913 mm. The distancebetween the at least two impellers, also known as impeller separation(D_(s)) is for the 20 000 l bioreactor, according to the invention,preferably at least 1200 mm and at most 1700 mm, more preferably theimpeller separation is 1498 mm. The impeller shaft diameter for the 20000 l bioreactor according to the invention is preferably at least 51 mmand at most 64 mm. If the 20 000 l bioreactor according to the inventionhas baffles, the length of the baffles is preferably at least 4000 mmand at most 4600 mm, more preferably the length is 4365 mm. The width ofthe baffles for the 20 000 l bioreactor, according to the invention, ispreferably at least 260 mm and at most 290 mm, more preferably the widthis 279 mm. The baffle clearance for the 20 000 l bioreactor, accordingto the invention, is preferably at least 20 mm and at most 35 mm, morepreferably the baffle clearance is 28 mm. The baffle height (H_(baffle))for the 20 000 l bioreactor according to the invention is preferably atleast 3600 mm and at most 4050 mm, more preferably the baffle height is3882 mm. The 20 000 l bioreactor according to the invention haspreferably at least one sparger, more preferably it has two spargers. Ifthe 20 000 l bioreactor according to the invention has two spargers oneis preferably a control sparger and one is preferably a ballast sparger.The control sparger for the 20 000 l bioreactor according to theinvention has preferably an orifice- or pore-size of at least 3 mm andat most 5 mm, more preferably the orifice- or pore-size is 4 mm. Theballast sparger for the 20 000 l bioreactor according to the inventionhas preferably an orifice- or pore-size of at least 5 mm and at most 7mm, more preferably the orifice- or pore-size is 6 mm. The orifice/porenumber of the control sparger for the 20 000 l bioreactor according tothe invention is preferably at least 230 and at most 270, morepreferably the orifice- or pore-number is 250. The orifice-pore-numberof the ballast sparger for the 20 000 l bioreactor according to theinvention is preferably at least 85 and at most 115, more preferably theorifice- or pore-number is 100. The sparger length (S_(L)) for thecontrol and/or the ballast sparger is preferably at least 500 mm and atmost 2000 mm, more preferably the sparger length is 1077 mm. The spargerto tank bottom clearance (S_(c)) of the 20 000 l bioreactor according tothe invention is preferably for the control and/or the ballast spargerat least 560 mm and at most 620 mm, more preferably the sparger to tankbottom clearance is 593 mm. The sparger to bottom impeller clearance(D_(c)−S_(c)) of the 20 000 l bioreactor according to the invention isfor the control and/or the ballast sparger preferably at least 300 mmand at most 340 mm, more preferably the sparger to bottom impellerclearance is 320 mm. The requirement to add ballast from a separatesparger, the ballast sparger, prevents dilution of oxygen or oxygenenriched DOT demand gas with the ballast gas. This ensures the bestoxygen transfer rate (OTR), as the oxygen concentration gradient of thebubbles emerging from the sparger is greatest. Secondly, the use of aballast sparger allows spargers to be located at different positions toavoid impacting DOT control on delivering desired ballast for pCO₂control. The ballast sparger can be independently designed from thecontrol sparger.

With the bioreactor design according to the invention, differentsubculture ratios can be performed. In a particularly preferredembodiment the subculture ratios performed are subculture ratios of atleast 1 in 5 (20% v/v) and at most 1 in 9 (11% v/v), more preferred 1 in5 (20% v/v) or 1 in 9 (11% v/v).

The invention also includes a method to cultivate and propagatemammalian cells, characterised in that at least one mammalian cell iscultivated under suitable conditions and in a suitable culture medium ina bioreactor according the invention.

Bioreactors according to the invention include all bioreactors having atleast two impellers and showing at least one feature or a combination ofdifferent features outlined above.

In the method according to the invention, the agitation rate of the atleast two impellers of the bioreactor is preferably at least 55 W/m³ andat most 85 W/m³. Preferably, air is sparged into the culture medium witha speed of at least 5×10⁻⁵ m/s, more preferably of at least 10×10⁻⁵ m/s.

In a particularly preferred embodiment of the present invention alkaliis added through two addition ports to distribute the alkali, which are,preferably spatially separated from each other. This ensures quickerblending of alkali in the event of long re-circulation time in the tank.CO₂ is preferably added via a control sparger.

Alkali and/or CO₂ are preferably used to regulate the pH of theculture-medium.

It is preferred that control and back-up probes be in the lower portring at 913 mm from tank bottom.

In a preferred embodiment of the present invention, the method accordingto the invention takes place in a bioreactor with a volume of 1000 l.The volume of the culture medium used in the method using a 1000 lbioreactor is preferably during the pre-inoculation 50 l to 250 l.During the post-inoculation the volume of the culture medium ispreferably at least 300 l and at most 960 l. In the pretransfer/harvestphase, the volume of the culture medium in the 1000 l bioreactor ispreferably at least 300 l and at most 960 l. The minimum operatingvolume (V_(min)) in a bioreactor with the volume of 1000 l according tothe invention is preferably between 80 l and 120 l, more preferably theminimum operating volume is 100 l, the maximum operating volume (V) ispreferably at least 900 l and at most 1100 l, the maximum operatingvolume is more preferably 1000 l. The minimum stirred volume ispreferably at least 230 l and at most 255 l, more preferably the minimumstirred volume is 245 l. The liquid height at the minimum operatingvolume (H_(min)) is in a bioreactor with a volume of 1000 l preferablyat least 210 mm and at most 240 mm, more preferably the liquid height atthe minimum operating volume is 228 mm. The liquid height at the maximumoperating volume (H_(L)) in a bioreactor with a volume of 1000 l ispreferably at least 1500 mm and at most 1900 mm, more preferably theliquid height at the maximum operating volume is 1764 mm. The minimumaspect ratio (H_(min)/T) is preferably at least 0.15 and at most 0.19,more preferably the minimum aspect ration is 0.17. The maximum aspectratio (H_(L)-T) for the bioreactor with a volume of 1000 l used in amethod according to the invention is preferably at least 1.8 and at most2.1, more preferably the maximum aspect ratio is 1.96. The freeboardvolume is preferably at least 270 l and at most 310 l, more preferablythe freeboard volume is 293 l. The freeboard height is preferably atleast 450 mm and at most 550 mm, more preferably the freeboard height is500 mm. The total straight height (H) is preferably at least 1900 mm andat most 2200 mm, more preferably the total straight height is 2045 mm.The height of the upper probe- or sample-ring is preferably at least 900mm and at most 1200 mm, more preferably the height of the upper probe-or sample-ring is 1093 mm. The height of the lower probe-sample ring ispreferably at least 152 mm and at most 286 mm.

In a preferred embodiment of the present invention, the method accordingto the invention takes place in a bioreactor with a volume of 4000 l.The volume of the culture medium used in the method using a 4000 lbioreactor is preferably during the pre-inoculation 1914 l to 3077 l.During the post-inoculation the volume of the culture medium ispreferably at least 2153 l and at most 3846 l. In thepretransfer/harvest phase, the volume of the culture medium in the 4000l bioreactor is preferably at least 2153 l and at most 3846 l. Theminimum operating volume (V_(min)) in a bioreactor with the volume of4000 l according to the invention is preferably between 1500 l and 2200l, more preferably the minimum operating volume is 1900 l, the maximumoperating volume (V) is preferably at least 3800 l and at most 4200 l,the maximum operating volume is more preferably 4000 l. The minimumstirred volume is preferably at least 1500 l and at most 1800 l, morepreferably the minimum stirred volume is 1654 l. The liquid height atthe minimum operating volume (H_(min)) is in a bioreactor with a volumeof 4000 l preferably at least 800 mm and at most 1200 mm, morepreferably the liquid height at the minimum operating volume is 1024 mm.The liquid height at the maximum operating volume (H_(L)) in abioreactor with a volume of 4000 l is preferably at least 1800 mm and atmost 2200 mm, more preferably the liquid height at the maximum operatingvolume is 2034 mm. The minimum aspect ratio (H_(min)/T) is preferably atleast 0.55 and at most 0.75, more preferably the minimum aspect rationis 0.63. The maximum aspect ratio (H_(L)-T) for the bioreactor with avolume of 4000 l used in a method according to the invention ispreferably at least 1.1 and at most 1.4, more preferably the maximumaspect ratio is 1.25. The freeboard volume is preferably at least 850 land at most 1250 l, more preferably the freeboard volume is 1039 l. Thefreeboard height is preferably at least 450 mm and at most 550 mm, morepreferably the freeboard height is 500 mm. The total straight height (H)is preferably at least 2000 mm and at most 2400 mm, more preferably thetotal straight height is 2252 mm. The height of the upper probe- orsample-ring is preferably at least 1200 mm and at most 1600 mm, morepreferably the height of the upper probe- or sample-ring is 1403 mm. Theheight of the lower probe- or sample-ring is preferably at least 500 mmand at most 550 mm, more preferably the height of the lower probe- orsample-ring is 531 mm.

In a preferred embodiment of the present invention, the method accordingto the invention takes place in a bioreactor with a volume of 20 000 l.The volume of the culture medium used in the method using a 20 000 lbioreactor is preferably during the pre-inoculation 13 913 l to 17 096l. During the post-inoculation the volume of the culture medium ispreferably at least 17 391 l and at most 19 231 l. In thepretransfer/harvest phase, the volume of the culture medium in the 20000 l bioreactor is preferably at least 20 000 l and at most 21 739 l.The minimum operating volume (V_(min)) in a bioreactor with the volumeof 20 000 l according to the invention is preferably between 9000 l and16 000 l, more preferably the minimum operating volume is 13 000 l, themaximum operating volume (V) is preferably at least 19 000 l and at most25 000 l, the maximum operating volume is more preferably 22 000 l. Theminimum stirred volume is preferably at least 8100 l and at most 8500 l,more preferably the minimum stirred volume is 8379 l. The liquid heightat the minimum operating volume (H_(min)) is in a bioreactor with avolume of 20 000 l preferably at least 2100 mm and at most 2500 mm, morepreferably the liquid height at the minimum operating volume is 2309 mm.The liquid height at the maximum operating volume (H_(L)) in abioreactor with a volume of 20 000 l is preferably at least 3550 mm andat most 3950 mm, more preferably the liquid height at the maximumoperating volume is 3777 mm. The minimum aspect ratio (H_(min)/T) ispreferably at least 0.70 and at most 0.99, more preferably the minimumaspect ration is 0.83. The maximum aspect ratio (H_(L)-T) for thebioreactor with a volume of 20 000 l used in a method according to theinvention is preferably at least 1.2 and at most 1.5, more preferablythe maximum aspect ratio is 1.35. The freeboard volume is preferably atleast 5750 l and at most 6500 l, more preferably the freeboard volume is6131 l. The freeboard height is preferably at least 900 mm and at most1100 mm, more preferably the freeboard height is 1000 mm. The totalstraight height (H) is preferably at least 3700 mm and at most 4100 mm,more preferably the total straight height is 3968 mm. The height of theupper probe- or sample-ring is preferably at least 2200 mm and at most2650 mm, more preferably the height of the upper probe- or sample-ringis 2411 mm. The height of the lower probe- or sample-ring is preferablyat least 880 mm and at most 940 mm, more preferably the height of thelower probe- or sample-ring is 913 mm.

For a bioreactor with a volume of 20 000 l the preferred seeding ratioused is 11% v/v (1 in 9 dilution) or 20% v/v (1 in 5 dilution), with apreferred feed application of 4% v/v to 25% v/v of the post-inoculationvolume. The post-inoculation volume in the 20 000 l bioreactor ispreferably adjusted for feed applications up to 15% such that after theaddition of all the feeds the final volume at harvest ends up at 20 000l. However, for feed applications greater then 15% v/v thepost-inoculation volume is preferably adjusted for a 15% v/v feed butfollowing the application of feeds the final pre-harvest volume will bea minimum of 20 000 l and a maximum 22 000 l. The 20 000 l bioreactor isexpected to hold a total of 20 000 l to 22 000 l at the end of a batch.

The bioreactor with a volume of 20 000 l is preferably operated in batchor fed batch mode for 10 to 15 days.

The invention also includes a bioreactor system for the cultivation ofmammalian cells characterised in that a) a first bioreactor with avolume of at least 500 l, preferably of at least 1000 l, is connectedwith b) a second bioreactor with a volume of at least 2000 l, preferablyof at least 4000 l, which has a volume greater than the first bioreactorand wherein the second bioreactor with a volume of at least 2000 l,preferably of at least 4000 l, is connected with c) a third bioreactoraccording to the invention having a volume of at least 10 000 l,preferably of at least 20 000 l, which has a volume greater than thesecond bioreactor.

In a preferred embodiment of the invention, the bioreactor system ischaracterised in that at least one of the bioreactors is a bioreactoraccording to the invention. More preferably, all of the bioreactors ofthe bioreactor system are bioreactors according to the invention.

Bioreactors according to the invention are in this context allbioreactors described in this description, in the examples and in theclaims.

The bioreactor system according to the invention is also calledbioreactor train or device.

The bioreactor train comprises preferably different bioreactors, whichare also called stage. The bioreactor with a volume of at least 500 l,preferably of at least 1000 l corresponds to stage N-3 and/or N-2. Thebioreactor with a volume of at least 2000 l, preferably of at least 4000l corresponds to stage N-1. The bioreactor with a volume of at least 10000 l, preferably of at least 20 000 l corresponds to stage N.

The design of the bioreactor train is based on the need to ensure ahomogenous environment with respect to process parameters such as pH,dissolved oxygen tension (DOT) and temperature, maintaining a well mixedcell suspension and blending nutrient feeds within the bioreactor. Thebioreactors of the bioreactor train preferably show geometricsimilarity. This allows a scale-down model to develop, for example at 12l laboratory scales or 500 l pilot scales. The bioreactors of the stagesN-3, N-2 and N-1 are used as seedbioreactors. Bioreactor of stage N isused as a productionbioreactor. The design of the seed- andproduction-bioreactors is preferably based on the same principles.However, some departures can be required to allow for flexibility inprocessing.

In a preferred embodiment of the invention, the aspect ratio H_(L)/T isat least 0.17 and at most 1.96.

In a preferred embodiment of the invention there is a furtherbioreactor, especially a 50 l bioreactor corresponding to stage N-4.

In a preferred embodiment of the invention, the N-4 bioreactor is aS-200 seed wave bioreactor or a 100 l stirred tank reactor

In a preferred embodiment of the invention, liquids, for example culturemedium, can be transported from one bioreactor to another bioreactor bypneumatic assisted flow or by peristaltic pumps.

The invention also includes a method to cultivate and propagatemammalian cells, characterised in that a) at least one mammalian cell iscultivated under suitable conditions and in a suitable culture medium ina first bioreactor with a volume of at least 500 l, preferably with avolume of at least 1000 l, b) the medium containing the cells obtainedby propagation from the at least one mammalian cell is transferred intoa second bioreactor with a volume of at least 2000 I, preferably with avolume of at least 4000 l, c) the transferred cells are cultivated inthe second bioreactor with a volume of at least 2000 I, preferably witha volume of at least 4000 l, d) the medium containing the cells obtainedin step c) is transferred into a third bioreactor with a volume of atleast 10 000 l, preferably with a volume of at least 20 000 l, and e)the transferred cells are cultivated in the third bioreactor with avolume of at least 10 000 l, preferably with a volume of at least 20 000l.

In a preferred embodiment of the invention, the method is characterisedin that at least one of the bioreactors used is a bioreactor accordingto the invention, more preferably all bioreactors used are bioreactorsaccording to the invention.

Bioreactors according to the invention are in this context allbioreactors described in this description, in the examples and in theclaims.

The bioreactor of step e) is preferably operated in batch or fed batchmode. The cells are cultivated in step e) preferably for 10 to 15 days.

Step a) is also called stage N-3 and/or N-2. Step c) is also calledstage N-1. Step e) is also called stage N.

Preferably the cultivation conditions in the bioreactors of steps a), c)and e) are the same. More preferably, the cultivation conditions in thebioreactors of steps a), c) and e) have a homogenous environment withrespect to process parameters such as pH, dissolved oxygen tension andtemperature. Preferably pH, dissolved oxygen tension and temperature inthe bioreactors of steps a), c) and e) are the same.

In a preferred embodiment of the invention, the seeding ratio after thetransfer steps b) and/or d) is at least 10% v/v, more preferably atleast 11% v/v (1 in 9 dilution) and at most 30% v/v, more preferably 20%v/v (1 in 5 dilution).

Preferably either the total medium or only a part of the medium aretransferred in steps b) and d).

Further preferred embodiments of the present invention are thesubject-matter of the sub claims.

The present invention is illustrated in more detail in the followingexamples and the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a bioreactor according to the invention. 1 is thebioreactor. 10 is the diameter of the tank (T). 20 is the total straightheight of the bioreactor (H). 30 is the base height of the bioreactor(H_(b)). 40 is the head height of the bioreactor (H_(h)). 50 is theliquid height at the maximum operating volume (Hp. 60 is the topimpeller diameter (D_(top)). 68 is the top impeller. 70 is the bottomimpeller diameter (D_(bottom)). 78 is the bottom impeller. 80 is theclearance between tank bottom and centre line of the bottom impeller(D_(c)). 90 is the impeller separation (D_(s)). 100 is the clearance ofthe top impeller below the liquid surface (D_(o)). 108 is a sparger. 110is the sparger to tank bottom clearance (S_(c)). 120 is the sparger tobottom impeller clearance (D_(c)−S_(o)). 128 is a baffle. 138 is a portlocated at the lower ring. 148 is a port located at the centre-line ofthe top impeller 68.

FIG. 2 shows a bioreactor system of the present invention. 111 is abioreactor with a volume of 1000 l. 11 is a bioreactor with a volume of4000 l. 1 is a bioreactor according to the invention with a volume of 20000 l.

EXAMPLES Example 1 20 000 l Bioreactor

The 20 000 l bioreactor is operated in batch and fed batch mode for 10to 15 days for the cultivation of mammalian cells. The mammalian cellsare kept in a homogeneous suspension by agitation via an impellersystem.

Vessel Geometry

The vessel geometry for the 20 000 liter bioreactor was determined by aniterative design basis in which the maximum working volume, freeboardstraight side distance, aspect ratio H_(L)/T and impeller to tankdiameter, D/T ratio are altered until an acceptable aspect ratio isachieved.

Bioreactor Aspect Ratio H_(L)/T

This critical design parameter allows characterisation of bioreactorgeometry. Tanks with higher aspect ratio offer longer gas residence timeallowing greater K_(L)a. However increased head pressure can cause buildup of soluble gases. Smaller aspect ratio H_(L)/T in tanks can lead toshorter gas residence time requiring greater gas flow for aerationresulting in greater foam build up. Impeller driven agitation toincrease K_(L)a is also limited by H_(L)/T as surface breakage andvortex creation will occur at lower impeller revolutions in a low aspectratio. Thus choice of aspect ratio is largely experience based with somethought on issues highlighted in table 1.

TABLE 1 Summary of effect of varying aspect ratio Process factor Highaspect ratio Low aspect ratio Radial mixing More effective Lesseffective Mixing time Higher Lower Oxygen transfer Determined bydissolved Determined by dissolved rate oxygen control oxygen control Gasflow rate Lower Higher Cell damage Less More Carbon dioxide Lesseffective More effective stripping Pressure variations Higher Lower Easeof scale up/ More difficult away from More difficult away from scaledown (access currently used aspect currently used aspect to scale data)ratios ratios Cleanability Not affected directly by Not affecteddirectly by aspect ratio aspect ratio Volume flexibility Less More

Table 2 describes the aspect ratios in the 20 000 liter bioreactor atvarious operating volumes during normal processing. The aspect ratioshave been tested at 500 liter scale and provided the superficial gasvelocity and power per unit volume are kept constant the K_(L)a remainsconstant.

TABLE 2 Key operating volumes and aspect ratios in the 20000 litrebioreactor Volume, L Liquid head, mm Aspect ratio, H_(L)/TPre-Inoculation 13913-17096 2458-2977 0.88-1.07 Post Inoculation17391-19231 3025-3325 1.08-1.19 Harvest 20000-21739 3451-3734 1.23-1.34Tank Diameter

The tank diameter is altered to obtain the optimal aspect ratio H_(L)/T.Changes to tank internal diameter (ID) are limited by acceptable aspectratio and plant footprint. The ID is 2.794 m.

Tank Height

Tank height is determined from the maximum operating volume, aspectratio H_(L)/T, freeboard straight side length, base and top platedesign. The final tank height is a compromise value determined fromvolumetric contingency for foam, plant height and impeller shaft length.The tank height from base to head tan line is 4.933 m.

Freeboard Height

The freeboard height is defined as the length of straight side above theliquid head when the bioreactor is filled to it's maximum operatingvolume. This is determined by taking into account the extent of:

-   -   Foam build up during operation.    -   Gas hold up at maximum allowed agitation and aeration.    -   Errors in metering liquid.

In absence of knowing the exact contribution of each with piloting theprocess at full scale an estimate is usually made. The amount offreeboard height is balanced with the desire to reduce the impellershaft length for a top-driven system, where extra length can complicatethe design and selection of available mechanical seals, the requirementfor steady bearing or stabilising impeller rings. A minimum freeboardheight of 1000 mm (or 6100 liter volumetric capacity or 28% v/v of themaximum operating volume) is therefore used.

Head and Base Plate

The selection of head and base plate design was made with aconsideration for desired mechanical strength, free draining cleandesign and fluid flow. Maintaining consistent plate design between scaledown and full scale will contribute towards maintaining geometricsimilarity. The base plate is of American Society of MechanicalEngineers Flanged and Dished (ASME F&D) design. The head-plate designaccommodates a manway or a flanged head plate to allow access/removal ofthe impellers.

Bioreactor Agitation Requirement

The agitation of the bioreactor is to achieve rapid mixing, maintainhomogeneity, maintain mammalian cells in suspension and gas bubbledispersion. The underlying issue with achieving the above objectives isminimising cell damage through shear forces originating from impellergeometry and eddies or vortices created behind the impeller blades. Acompromise of the above objectives can be achieved by selection of anappropriate impeller type.

Bottom Versus Top Driven Impeller Shaft

The decision to drive the agitator shaft from the top or the bottom ofthe bioreactor is important and is determined following a review of anumber of issues highlighted in table 3.

TABLE 3 Key design issues for selection of top versus bottom entry ofimpeller shaft Top entry Bottom entry Shaft Length Long Short ShaftWeight High Low Shaft Diameter Larger Smaller Impeller shaft on-siteGreater plant Less plant height installation and removal height forservicing and repair Exposure of cell culture to No exposure Exposuremoving and stationary seal faces ¹Pressurization between Lower Higherdue to the liquid seal and vessel head Seal Lubricant leakage rate LowerHigher Base plate Design Simple Complex Sparger to tank bottomUnrestricted Restricted positioning CIP validation Simple Complicated bysub- merged mechanical seal Scale up and scale down Consistent withInconsistent with lab and consistency lab and pilot scale pilot scale¹Pressure differential between seal and bioreactor critical forlubrication and cooling.

Top-entry impeller shafts tend to be longer than bottom-entry, whichresults in the shaft being heavier and larger diameter. Additionally theshaft length together with the inherent clearance between the two facesof the mechanical seal may dictate the requirement for steady bearingsor stabilising ring to prevent excessive “shaft wobble”. Service andmaintenance are affected by the available space around the agitator,gearbox and seal assembly, and on-site shaft installation and removal islimited by plant height.

The protrusion of the seal and impeller shaft at tank bottom restrictsthe placement of the sparger near the tank bottom. This dimensionaffects the tank hydrodynamics and therefore its amenability to changeis important in specifying an optimal design.

The downwards load of down pumping impellers together with the liquidhead have an accumulative greater load (compared to up pumping ortop-entry shaft) between the moving and stationary faces of the sealresulting in greater wear of the seal faces. Furthermore loss of overpressure in the condensate line supplying the seal can result in theculture seeping into the seal. This makes the subsurface seal a lesssanitary design.

The submerged seal complicates the design of a free draining bioreactorby compromising the position of the harvest drain valve. Secondly thediameter of the harvest nozzle may be restricted thus restricting theflow rate of harvest stream. Therefore a top entry impeller shaft isused in the 20 000 liter bioreactor.

Baffles

The baffle requirement for centre mounted impeller is critical toprevent vortex formation. The critical issues related to baffles arebaffle number, baffle width (W), baffle length (H_(baffle)) and baffleto tank wall clearance (W_(c)).

The recommendation for four equally spaced baffles that are 0.1×T or 279mm wide 1.1×H−H_(h), or 3882 mm tall and have a baffle to tank wallclearance, W_(c) of 0.01×T or 28 mm.

The thickness of baffle is not specified but the thickness needs toensure rigidity to the radial component of the fluid flow. Additionallythickness needs to ensure the baffle plates are not warped during SIPthereby affecting the baffle to tank wall clearance.

Impeller Type

High shear, such as Rushton (or Rushton-type), impellers offer highpower dissipation for gas dispersion but lack in axial flow necessaryfor mixing and homogeneity. Additionally, agitation from high shearimpellers suffers from dangers of excessive cell damage.

Table 4 shows the impellers tested at lab scale (12.2 liter) that gaveequivalent hydrodynamic and cell growth performance. The hydrofoil ismounted above the high solidity pitched blade impeller.

The Lightnin A310 and A315 at the D/T ratio described in table 4 areused in the bioreactor.

TABLE 4 Impeller types short-listed for scale down study Impellers D/Tratio ¹N_(p)/²N_(q) Vendor Description A310 0.44 0.30/0.56 LightninThree bladed hydrofoil design A315 0.46 0.75/0.73 Lightnin Four pitched-bladed high solidity impeller SC-3 0.40 0.90/0.90 Chemineer Three bladedhydrofoil design 3HS39 0.46 0.53/0.58 Philadelphia Four pitched- Mixersbladed high solidity impeller ¹N_(p) is characteristic impeller powernumber. It is a measure of an impeller efficiency to impart the kineticenergy of the rotating impeller blades to the fluid. It is important inquantifying the gas dispersion ²N_(q) is characteristic impeller flownumber. It is a measure of pumping ability of the impeller and isimportant in quantifying fluid bulk movement.Impeller to Tank Diameter, D/T Ratio

The diameter for axial flow impellers is recommended to be less than0.5×T. A diameter greater than this results in disruption in axial flow,hence poor agitation and aeration.

Power dissipation into the bioreactor and Reynold's number also need tobe sufficiently high to maintain a turbulent (loaded) regime. Thereforethe selection of impeller diameter is a compromise between choosinglarge enough diameter to ensure adequate homogeneous mixing withoutexceeding the hydrodynamic characteristics of the bioreactor. Theseinclude throttling axial flow, insufficient power dissipation, exceedingupper limits of impeller tip speed and creation of poorly mixed laminarzone.

Once a diameter is selected, than maintaining constant D/T ratio iscritical between scale down pilot vessels in order to maintain thecentral assumption of scale studies—that of maintaining geometricsimilarity.

The K_(L)a scale up correlation at 12.2 liter has been determined forthe four impellers at the D/T ratios shown in table 4. From a geometricsimilarity standpoint A310 diameter of 1.229 m (D/T of 0.44) and A315diameter of 1.285 m (D/T of 0.46) is recommended. However a manwaydiameter can restrict the largest impeller diameter that can beinstalled and removed to 1.219 m. Therefore A310 and A315 to be 1.219 mdiameter are used thereby keeping with ease of impeller installation andremoval and maintaining close to the geometric similarity proposed inscale down study.

The Impeller Clearance, D_(c) and Spacing, D_(s)

The spacing between impellers in a bioreactor with multiple impellers isan important dimension to consider. For a bioreactor with dual Rushtonturbine (radial flow) the ungassed power consumption is equivalent to asingle impeller when the dual impeller are spaced less then 0.5×D alongthe shaft. At a spacing of 2×D the power consumption becomes adductive.Thus efficiency of the impeller is reduced when the impeller spacingbecomes less then 0.5×D and the requirement for multiple impellersbecomes unnecessary. It is important to note that impeller spacing alsoimpacts on the potential of creating dead zones (poorly mixed zones)within the bioreactor.

An additional constraint on the choice of impeller spacing is discreteworking volumes required within the bioreactor.

The impeller spacing, D_(s), of 1.229×D_(bottom) (1498 mm) allows bothimpellers to remain submerged at the lowest post-inoculation volume of17392 liters with liquid head above the upper impeller, D_(o), of0.5×D_(top) (615 mm) and off bottom clearance, D_(c), of 0.75×D_(bottom)(913 mm).

Table 5 highlights volumes that will form liquid surfaces or lowerliquid cover, above the impellers. Agitation needs to be modified toavoid foaming at these critical volumes.

TABLE 5 Key operating volumes that cause interaction with impellers andliquid surface Interaction Volume, L Potential Operation Submerge topimpeller with 17399 Minimum post inoculation volume 17391 L 0.5D_(A310)liquid cover Liquid surface touching top 13973 Pre-inoculation volume of13913 L liquid surface edge of top impeller breakage Liquid surfacetouching 13283 Bolus addition of pre-inoculation medium will pass bottomedge of top impeller through this liquid head Submerge bottom impeller8381 Bolus addition of pre-inoculation medium will pass with 0.5D_(A315)liquid cover through this liquid head Liquid surface touching top 5592Bolus addition of pre-inoculation medium will pass edge of bottomimpeller through this liquid head Liquid surface touching 3291 Bolusaddition of pre-inoculation medium will pass bottom edge of bottomthrough this liquid head impeller ⁽¹⁾Minimum operating volume with lowerimpeller submerged is 8379 litres and minimum operating volume with bothimpellers submerged is 17399 litres ⁽²⁾The operating volume range is13913 to 21739 litres.Clearance of Top Impeller Below Liquid Surface, Do.

The breakage of the impeller blade above liquid surface is undesirableas this will make the flow and power dissipation of the impellerineffective. In addition it will create unknown K_(L)a values due tosignificant surface entrapment of headspace gas into the fluid andexcessive foam. D_(o) is 0.3×D for radial flow impellers and 0.5×D foraxial flow impellers such as A310. However as D_(o) approaches 2×D theimpeller provides gentle blending duty. This is acceptable for theproduction bioreactor application as K_(L)a study has shown thatbioreactor K_(L)a is influenced mostly by the bottom A315 impeller andthe top A310 impeller contributes to bulk mixing.

As a result of setting D_(c) and D_(s) at values D_(o) is maintained atan optimal range for the duration of operation of the productionbioreactor. During the course of a batch the liquid cover above the topimpeller will change from 0.5×D_(A310) and 1.08×D_(A310). The liquidcover above the top impeller will increase as the bioreactor is fednutrient feeds and alkali to maintain constant pH. Table 6 shows a rangeof liquid cover above the top impeller for a range of operating volumes.

TABLE 6 Key operating volumes and the liquid cover above top impeller,Do Cylinderical height, H Do, mm Do as ratio Operating volume, L mm(inches) (inches) of D_(A310) Pre-Harvest, 21739 L 3252 (128″) 1324(52″)  1.08D_(A310) Pre-Harvest, 20000 L 2968 (117″) 1040 (41″) 0.85D_(A310) Post-Inoculation, 19231 L 2843 (112″) 915 (36″)0.74D_(A310) Post-Inoculation, 17391 L 2543 (100″) 615 (24″)0.5D_(A310)  Pre-Inoculation, 15385 L 2215 (87″)  287 (11″) 0.23D_(A310)Pre-Inoculation, 13913 L 1973 (78″)  45 (2″) 0.04D_(A310) ⁽¹⁾Off bottomimpeller clearance, Dc = 913 mm (0.75D_(A315)), Impeller separation, Ds= 1498 mm (1.229D_(A315)), tank ID of 2794 mm and height of ASME F&Dbase plate, H_(h) = 483 mm ⁽²⁾D_(o) = H − D_(s) − (D_(c) − H_(h))Agitation Rate—Rpm, P/V and Tip Speed

Table 7 below specifies the agitation rate for the 20 000 literbioreactor. The bioreactor is agitated typically at 20-260 W/m³,preferably at 55-85 100 W/m³. The agitation strategy is being developedduring the 500 liter pilot fermentations. The agitation rate of 0 to80±1 rpm is therefore used as an operational range.

TABLE 7 Agitation rate for the 20000 L bioreactor Power per unitAgitation rate, rpm volume, W · m⁻³ Tip Speed, m/s Pre- Typically 28-30can Typically 20 can 1.8-1.9 inoculation be higher be higher Post-Typically 56 can Typically 103, can 3.6 can be up inoculation be up to80 be up to 260 to 5.1 until harvestMechanical Seals Specification

For bioreactor all seals are to be double mechanical seals with amaximum “run out” or wobble tolerance of 0.2 mm. Three types wereconsidered; these include:

-   -   Wet seal lubricated with sterile condensate.    -   Dry seal lubricated with sterile gas such as N₂ or CA.    -   Non lubricated or floating seal that are uni-rotational.

All mechanical seals are recommended to be serviced on an annual basis.This requires the removal of seal from the bioreactor and sending theseal assembly to the vendor. Therefore the design must consider ease ofroutine maintenance.

The dry type seal (John Crane—5280D type) will produce 3 g per year ofshedding (seal face and seal seat material) composed of resinimpregnated carbon. This is based on continuous 24 hour operation over ayear. The amount of shedding for the wet seal is significantly less.Therefore a wet condensate-lubricated seal is adopted for all bioreactordouble seals.

Bioreactor Aeration Requirement and Gassing Strategy

The aeration duty of the 20 000 liter bioreactor is governed by:

-   -   K_(L)a requirement.    -   DOT control strategy.    -   pCO₂ control/stripping strategy.    -   Use of sintered or fluted spargers.

The 20 000 liter bioreactor is designed to provide K_(L)a values of upto 20 h⁻¹ for processes with oxygen uptake rates of 5 mmol×L⁻¹×h⁻¹. Thebioreactor design needs to be flexible enough to allow cultivation ofprocesses reaching 20×10⁶ cells×mL⁻¹.

The aeration requirement can be achieved by a number of differentapproaches. However the use of a fluted sparger with air and oxygenenrichment to make up any deficit in oxygen transfer rate (OTR) duringpeak oxygen demand was used. The advantages of this approach are:

-   -   Easier CIP and SIP validation of fluted sparge design.    -   Larger air throughput to aid dissolved CO₂ stripping.    -   Reduced operating cost through the avoidance of purchase of        single use sintered elements.

The disadvantages of the approach selected above also need to beconsidered. These include:

-   -   Inherent lower K_(L)a for the low power number impellers        selected.

Therefore the bioreactor aeration design must have the flexibility to bemodified to meet the desired K_(L)a.

Table 8 describes the gassing requirements for the 20 000 literbioreactor. The gas flow rates were scaled up on constant superficialgas velocity.

Two spargers are used. The main or “DOT control” sparger supplied bydual range clean air, mass flow controller (MFC) and oxygen MFC with gasflow metered via a DOT control loop and a CO₂ MFC metering gas via theacid pH control loop. The dual range MFC's are used to achieve preciseflow control at the extreme ends of the desired operating ranges.

The second or “ballast” sparger is supplied by a CA MFC to whichnitrogen is also supplied. It was measured that early DOT controlrequires small nitrogen ballast to assist in early DOT demand and lowerthe DOT to set point. The ballast sparger also meters ballast air tofacilitate stripping out excess pCO₂.

The headspace purge is used to allow removal of CO₂ and oxygen from theheadspace. This is to facilitate better pH and pCO₂ control and dilutionof high oxygen blend prior to exhausting to environment. The ability tovary headspace flow rate allows design of gassing strategy for variousprocesses requiring different blends of oxygen enrichment and controlpoint pCO₂.

TABLE 8 Gas flow rate and MFC operating ranges for the 20000 litre bio-reactor Gas Operating range Comments Head Space¹ 1.) Clean air 1.)0-1000 1.) Head space purging of CO₂ and    SLPM    O₂ 2.) Nitrogen 2.)Utility rated 2.) For rapid DOT probe zeroing 3.) Helium 3.) Utilityrated 3.) Tank integrity testing DOT control Sparger 1.) Clean air² 1.)10-500 1.) Gas flow under DOT control    SLPM 2.) Oxygen 2.) 10-100 2.)Gas flow under DOT control Carbon dioxide³    SLPM 3.) 2-150 SLPM 3.)Gas flow under pH control Ballast Sparger 1.) Clean air 1.) 20-500 1.)Variable ballast for dCO₂    SLPM    stripping 2.) Nitrogen⁴ 2.) 20-5002.) Early DOT control by variable    SLPM    flow ¹The air and nitrogengas flow into headspace enters via a bypass for post SIP tankpressurisation. ²Clean air gas flow operating range achieved by a dualCA MFC at 5-50 SLPM and 50-500 SLPM respectively. ³CO₂ gas flowoperating range achieved by a dual CO₂ MFC at 2-30 SLPM and 30-150 SLPMrespectively. ⁴Both air and nitrogen gas flow metered from a common CAMFC.

The bioreactor ports for sparger installation are designed to fit pipedesign of diameter of 51 mm. The position of port should allow theplacement of control sparger (D_(c)−S_(c)) at a distance of 320 mm belowthe bottom edge of the lower impeller and no greater 593 mm from tankbottom (S_(c)).

This results in a S_(c) value of 593 mm or (0.65×D_(c)) and this fallsoutside the acceptable range of 0.2×Dc to 0.6×Dc. However hydrodynamictrials in 500 l suggest S_(c) clearance of 0.41 to 0.71×D_(c) has noimpact on measured K_(L)a.

A separate port for the installation of the ballast sparger was alsobuilt. The position of this port allows the placement of ballast spargerat a distance of 320 mm, (D_(c)−S_(c)) below the bottom edge of thelower impeller and no greater then 593 mm from tank bottom (S_(c)). Therequirement to add ballast from a separate sparger is due to threereasons:

-   -   Firstly, it prevents dilution of oxygen or oxygen enriched DOT        demand gas with the ballast gas. This ensures the best OTR, as        the oxygen concentration gradient of the bubbles emerging from        the sparger is greatest.    -   Secondly, it allows ballast sparger to be located at a different        position from DOT control sparger to avoid impacting DOT control        on delivering desired ballast for pCO₂ control.    -   Thirdly, the ballast sparger can be independently designed from        the DOT control sparger.

The calculation of hole size and number of holes is iterated until thetarget Reynold's number, Re of gas emerging from holes is <2000 and theSauter mean diameter for a bubble is 10-20 mm during chain bubbleregime. Table 9 shows the key specifications for the control and ballastsparger for the 20 000 liter bioreactor.

TABLE 9 Design specification for the 20 000 litre bioreactor spargersDOT control Ballast Parameter sparger sparger Gas flow, SLPM 850 500Number of sparge holes 250 100 Orifice diameter, d_(o), m 0.004 0.006Gas flow, m³ · s⁻¹ 1.42E−02 8.33E−03 Orifice area, m² 1.26E−05 2.83E−05Total orifice area, m² 3.14E−03 2.83E−03 Density of air, Kg · m⁻³ 1.1661.166 Viscosity, Nm · s⁻² 1.85E−05 1.85E−05 Sauter mean diameter,d_(vs), mm 16.34 19.06 (d_(vs) = 1.17 V_(o) ^(0.4) d_(o) ^(0.8)g^(−0.2)) Gravitational acceleration, g m · s⁻² 9.807 9.807 Densitydifference, Kg · m⁻³ 1048.834 1048.834 Reynold's number, >2000 jettingregime 1139 1117 Gas velocity at sparger, V_(o), m/s 4.51 2.95 Spargerlength, S_(L), m 1.077 1.077 Combined length to drill required holes, m1.000 0.6 Number of rows to fit required holes in 2 1 length S_(L)Sparger to tank bottom clearance, Sc, m 0.593 0.593 Sparger to bottomimpeller clearance, Dc-Sc, m 0.320 0.320

A ring sparger of 0.8×D_(bottom) (80% diameter of bottom A-315 impellerdiameter) is used to distribute the holes under the blades and not theimpeller hub. However the CIP and installation of this configuration isdifficult. Therefore selection of sparger geometry that permitsdistribution of the desired number of holes in a manner that isconsistent with best to distribute the holes and sanitary design can beused also.

As an option a crescent rather then straight pipe is explored. Thecurvature of the crescent is 0.8×D_(bottom). In order to aidinstallation and removal from side ports of the bioreactor the crescentcircumference is 240° of the complete circumference of 0.8×D_(bottom)ring, this is 1077 mm.

The DOT control sparger is 1077 mm long and has a 51 mm diameter. Theholes have a 4 mm diameter. A total of 250 holes divided into 2 rows(2×125) at 45° from the dorsal (vertical) are used. Drain holes of 4 mmdiameter on both ends of the sparger are drilled on the ventral side ofthe sparger to aid free CIP drainage of the sparger.

The ballast sparger is 1077 mm long and of 51 mm diameter and has atotal of 100 6 mm diameter holes in a single dorsal row. Drain holes of4 mm diameter on both ends of the sparger are drilled on the ventralside of the sparger to aid free CIP drainage of the sparger.

Position of Probes, Addition and Sample Ports

The probe ring position must be placed in a well-mixed representativeregion of the bioreactor. Additional considerations included workingvolume range and ergonomic operations. The location of probe ports,sample valve and addition points were considered together to avoidtransitory spikes. Furthermore the position of the sample valve withrespect to controlling probes needs to permit accurate estimation ofoff-line verification of the measured process parameter. This is shownin table 10.

TABLE 10 Probe, addition and sampling port specification for the 20000litre bioreactor ²Diameter, mm ¹Position, mm Probe/Port Location(inches) (inches) Rational Temperature (main) Lower ring 38.1 (1.5″) 1.)913 (36″) In the plane of 2.) 30° centre-line of bottom impellerTemperature Lower ring 38.1 (1.5″) 1.) 913 (36″) In the plane of(backup) 2.) 170° centre-line of bottom impeller pH (main) Lower ring38.1 (1.5″) 1.) 913 (36″) In the plane of 2.) 10° centre-line of bottomimpeller pH (backup) Lower ring 38.1 (1.5″) 1.) 913 (36″) In the planeof 2.) 20° centre-line of bottom impeller DOT (main) Lower ring 25(0.98″) 1.) 913 (36″) In the plane of 2.) 150° centre-line of bottomimpeller DOT (backup) Lower ring 25 (0.98″) 1.) 913 (36″) In the planeof 2.) 160° centre-line of bottom impeller pCO₂ (spare) Lower ring 50.8(2″)tbd 1.) 913 (36″) In the plane of 2.) 20° centre-line of bottomimpeller Biomass (spare) Lower ring 50.8 (2″) 1.) 913 (36″) In the planeof 2.) 160° centre-line of bottom impeller Spare probe port Lower ring25 (0.98″) 1.) 913 (36″) In the plane of (DOT-type) 2.) 150° centre-lineof bottom impeller Spare probe port Lower ring 38.1 (1.5″) 1.) 913 (36″)In the plane of (pH-type) 2.) 10° centre-line of bottom impeller Samplevalve (main) Lower ring 12.7 (0.5″) 1.) 913 (36″) NovAseptic 2.) 40°type Sample valve Lower ring 12.7 (0.5″) 1.) 913 (36″) NovAseptic(backup) 2.) 50° type Alkali addition 1 - Lower ring 50.8 (2″) 1.) 913(36″) Diametrically Tank 1 2.) 190° opposite pH probes Alkali addition2 - Centre-line 50.8 (2″) 1.) 2411 (95″) Diametrically Tank 1 of upper2.) 190° opposite pH impeller probes Continuous feed 1 - Lower ring 50.8(2″) 1.) 913 (36″) Diametrically Tank 2 2.) 200° opposite pH probesContinuous feed 2 - Lower ring 50.8 (2″) 1.) 913 (36″) DiametricallyTank 3 2.) 210° opposite pH probes DOT control sparger N/A 101.6 (4″)1.) 593 (23″) Diametrically orifice 2.) 0° opposite ballast spargerBallast sparger orifice N/A 101.6 (4″) 1.) 593 (23″) Diametrically 2.)180° opposite control sparger Overlay gas Head plate 101.6 (4″) 1.) N/ADiametrically 2.) 135° opposite vent out Exhaust vent out Impeller 50.8(2″) 1.) N/A Diametrically flange 2.) 315° opposite overlay plate gas inHarvest valve Base plate 76.2 (3.0″) 1.) N/A NovAseptic 2.) Centre typeto allow free draining Antifoam addition Head plate 50.8 (2″) 1.) N/ALiquid surface/ 2.) 170° 0.25T from tank centre Shot feed 1 - LS1 Headplate 50.8 (2″) 1.) N/A Liquid surface 2.) 190° Shot feed 2 - GlucoseHead plate 50.8 (2″) 1.) N/A Liquid surface shot 2.) 180° Small add -Spare Head plate 50.8 (2″) 1.) N/A Liquid surface - 2.) 200° directedinto vessel wall Media inlet Head plate 101.6 (4″) 1.) N/A Nozzledirected 2.) 310° onto vessel wall Inoculum transfer Head plate 101.6(4″) 3.) N/A Nozzle directed from 4000 L to 4.) 320° onto 20000 L vesselwall CIP - Spray ball Impeller 76.2 (3″) 1.) N/A CIP'ing of flange 2.)270° highest point plate CIP - Spray ball Head plate 76.2 (3″) 5.) N/AAs per CIP 6.) 60° design CIP - Spray ball Head plate 76.2 (3″) 1.) N/AAs per CIP 2.) 180° design CIP - Spray ball Head plate 76.2 (3″) 1.) N/AAs per CIP 2.) 300° design Pressure indicating Head plate 38.1 (1.5″)1.) N/A As per vessel transmitter (PIT) 2.) 60° vendor design Pressuregauge Head plate 38.1 (1.5″) 1.) N/A As per vessel 2.) 50° vendor designRupture disc Head plate 101.6 (4″) 1.) N/A As per vessel 2.) 280° vendordesign Spare nozzle Head plate 101.6 (4″) 1.) N/A As per vessel 2.) 160°vendor design Sight glass Head plate 101.6 (4″) 1.) N/A As per vessel2.) 70° vendor design Light glass Head plate 76.2 (3″) 1.) N/A As pervessel 2.) 75° vendor design Manway Head plate 457.2 (18″) 1.) N/APersonnel 2.) 90° entry Agitator head/flange Head plate 1320.8 (52″) N/AEntry/removal Impeller shaft and Agitator 304.8 (12″) N/A Entry/removalseal manway head/ flange ¹Measured from the tangential line of the baseplate. Degrees pertain to plane of clockwise rotation. ²Diameter ofnozzle at bioreactor.Addition Ports, Surface and Sub-Surface

The need to determine addition ports that terminate at liquid surfaceand those that are subsurface was determined by operational scenariosand the effects of feed strategy on process control.

Currently the protein free process has two continuous feeds that need tobe discharged in well-mixed area of the bioreactor. Additional provisionfor glucose and an “LS1-type” shot addition is also integrated in thewell mixed region. The foam is controlled by surface addition of 1 in 10diluted C-emulsion. The inoculation of seed into the pre-inoculationbioreactor is served by avoiding build up of foam which will arise asthe culture is dropped onto the surface of the medium. Following portswere designed:

-   -   Six surface additions with media inlet, inoculum inlet, one        small addition inlet directed into the wall of the vessel while        the others dropped onto the liquid surface away from the tank        wall.    -   Four subsurface additions comprised of inlets from the two feed        tanks and bi-level inlet from the alkali tank.        Sample Ports

The sample port design allows a representative sample to be taken fromthe bioreactor. Therefore any residual material must be as small aspossible. The samples taken are used to determine off line checks fordissolved gases, pH, nutrients and biomass concentration. The orifice ofthe port opening is large enough to prevent sieving causing biomassaggregates to be retained. The 2 mm orifice NovaSeptum sampling devicewas used. However this has to be balanced with the desire to keepresidual volume of the port low. The port needs to be positioned in awell-mixed zone adjacent to the probes that need to be verified byoff-line checks and will be determined via nozzle position (see table10).

Add Tanks

In order to reduce cost and time the add-tanks supplying the bioreactorare of modular design. The production bioreactor has three 2500 liternominal volumes add tanks. The add tanks are filled at 25 l/min. Theflow rate of feeds from the add tanks to the bioreactor is controlled at0.2 to 1.0 milliliters of feed per liter of post-inoculation bioreactorvolume per hour (ml/l/h). It is expected that feed rate is controlled at±5% of set point.

The production bioreactor is serviced by three 1372 mm ID by 1880 mm addtanks. These tanks have the capability to be cleaned and sterilisedindependently and together with the production bioreactor.

Manway

Access into the bioreactor is required for certain service operations.Access can be gained by considering a flanged head plate orincorporation of a manway into the head plate. The need for access intothe bioreactor is for:

-   -   Installation of impellers.    -   Installation and replacing of impeller and impeller shaft.    -   Installation and replacing of mechanical seal.    -   Service of vessel furniture.    -   Potential modification of sparger position to obtain desired        hydrodynamic characteristics.

The size of the manway must be sufficient to allow access for the aboveobjectives. The manway used was of sufficient diameter to allow theremoval of two impellers of 1219 mm diameter.

Volume Measurement

The design ensures that any sensor gives sufficient precision in volumemeasurement around the operating range.

The volume measurement in bioreactor is able to measure a range of 13000 to 25 000 liters. The sensor sensitivity needs to be at least 0.5%of full span.

Volume measurement in the feed add-tanks and alkali tank is able tomeasure 0 to 2200 and 2500 liters respectively. The sensor sensitivityneeds to be at least 0.2% of full span. This will permit hourlyverification of feed flow rate at the minimum flow rate of 0.2 ml/l perhour or 3.5 l per hour by measuring the volume decrease in the addtanks.

Bioreactor Temperature Control

The medium is brought to operating temperature and pH by processcontrol. This is achieved by “gentle” heating of the jacket (avoid hightemperature at vessel wall). The temperature control range duringoperation is 36 to 38° C. with an accuracy of ±0.2° C. at set point.

Jacket

The bioreactor jacket area is specified with the followingconsiderations in mind:—

-   -   Steam sterilisation at 121-125° C.    -   Warming up of medium from 10° C. to 36.5° C. in <2 h.    -   All points within the bioreactor must reach ±0.2° C. of set        point, typically 36.5° C., as measured by thermocouples.    -   Chilling of medium from 36.5° C. to 10° C. in <2 h.        Bioreactor pH Control

The process pH is monitored and controlled with probes connected via atransmitter to a DCS based process controller. The process is becontrolled by addition of CO₂ to bring the pH down to set point andaddition of alkali to bring pH up to set point. pH is controlled at±0.03 of set point.

Alkali is added through two addition points to distribute the alkali.This ensures quicker blending of alkali in the event of longre-circulation time in the tank. The CO₂ is added via the controlsparger.

Control and back-up probes are located in the lower port ring at 913 mm(see table 10) from tank bottom. Additionally the pH probes are locateddiametrically opposite the alkali addition points into the bioreactor.

Bioreactor DOT Control

Dissolved oxygen is monitored and controlled with polarographic DOTprobe. The DOT set point maintained by sparging:

-   -   Initial N₂ ballast and/or air on demand.    -   Air ballast with air on demand.    -   Air ballast with oxygen on demand.    -   Reversing gas usage once oxygen demand decreases.

Cascade DOT control allows DOT set point to be maintained throughchanges in the ballast and demand gas in conjunction with ramping ofagitator speed.

In order to control pCO₂ the ballast required to strip out excess dCO₂impacts DOT control. Therefore the DOT control is considered togetherwith pCO₂ control for those processes where metabolic CO₂ is liberated.DOT is controlled at ±2% of set point. Control and back-up probes arelocated in the lower port ring at 913 mm from tank bottom.

Bioreactor Dissolved CO₂ Control

The process dCO₂ is monitored with an pCO₂ probe and excess dCO₂ isstripped by gassing CA through the ballast sparger. The optimal positionfor this probe is close to the pH probes.

Feed Addition Control

The feeds (SF22 and amino acid) are high in pH and osmolality. Thereforebolus additions need to be avoided to maintain good pH control. Howeverthe control of desired flow rates (±5% of set point) is technicallychallenging. Therefore an addition strategy that encompasses point ofaddition with delivery mode avoids the circulation of feed bolus andpotential variations of pH control.

Therefore the point of addition is in the plane of the centre-line ofthe bottom impeller that is 913 mm from tank bottom to assist in therapid blending of feed bolus.

Antifoam Addition Control

Antifoam (C-emulsion) addition is added as required to maintain thebioreactor liquid surface free of foam. A working stock of 1 in 10diluted C-emulsion can be dosed on the liquid surface. The antifoamsuspension is continuously agitated in the storage container to preventpartitioning. It is important to dose the antifoam close to the centreof the tank to diminish the effects of the radial component of the fluidflow carrying the antifoam to the tank walls where it will adhere.Therefore the addition point is 0.25×T toward the tank centre or 699 mmfrom tank centre.

Example 2 4000 Liter Bioreactor

Vessel Geometry

The vessel geometry for the 4000 liter bioreactor was determined by aniterative design basis in which the maximum working volume, freeboardstraight side distance aspect ratio (H_(L)/T) and impeller to tankdiameter ratio (D/T) are altered until an acceptable aspect ratio isachieved.

Bioreactor Aspect Ratio H_(L)/T

Table 11 describes the aspect ratios in the 4000 liter bioreactor at thevarious operating volumes during normal processing. These aspect ratiosarise from the selection of tank ID and the operating volume required.From a processing perspective the mixing requirements at the threeoperating conditions are different. During pre-inoculation stage thebioreactor mixing is important to allow medium to equilibrate withminimal K_(L)a requirement. However for post-inoculation andpre-transfer stages both mixing and K_(L)a are important considerations.Therefore both these features were tested at the aspect ratio range.

TABLE 11 Key operating volumes and aspect ratios in the 4000 litrebioreactor Volume, L Liquid head, mm Aspect ratio, H_(L)/TPre-Inoculation 1.) 1914 1.) 1031 1.) 0.63 2.) 2782 2.) 1448 2.) 0.893.) 3077 3.) 1590 3.) 0.98 Post Inoculation & 1.) 2153 1.) 1146 1.) 0.70Pre-transfer 2.) 3478 2.) 1783 2.) 1.10 3.) 3846 3.) 1960 3.) 1.21Tank Diameter

The tank diameter was altered to obtain the optimal aspect ratioH_(L)/T. Changes to tank internal diameter are limited by acceptableaspect ratio and plant footprint. The tank ID is 1626 mm.

Tank Height

Tank height is determined from the maximum operating volume, aspectratio H_(L)/T, freeboard straight side length, base and top platedesign. The final tank height is a compromise value determined fromvolumetric contingency for foam, plant height and acceptable impellershaft length. The head to base tan line height is 2817 mm.

Freeboard Height

The freeboard height of 500 mm (1039 liter or 27% v/v of the maximumoperating volume) is used for this seed bioreactor.

Head and Base Plate

The base and head plate design is a ASME F&D design for this seedbioreactor.

Bioreactor Agitation Requirement

The agitation of the bioreactor is to achieve rapid mixing, maintainhomogeneity, maintain mammalian cells in suspension and gas bubbledispersion. The underlying issue for achieving the above objectives isminimising cell damage through shear forces originating from impellergeometry and eddies or vortices created behind the impeller blades. Acompromise of the above objectives was achieved by selection of anappropriate impeller type.

Bottom Versus Top Driven Impeller Shaft

The motor drive is top mounted for the benefits already highlighted.

Baffles

The baffle requirement for centre mounted impeller is critical toprevent vortex formation. The critical issues related to baffles arebaffle number, baffle width (W), baffle length (H_(baffle)) and baffleto tank wall clearance (W_(c)).

Four equally spaced baffles that are 0.1×T or 163 mm wide 1.1×H−H_(h) or2195 mm tall and have a baffle to tank wall clearance, W_(c) of 0.01×Tor 16 mm were used.

The thickness of the baffles is not specified but the thickness needs toensure rigidity to the radial component of the fluid flow. Additionallythickness needs to ensure the baffle plates are not warped during SIPthereby affecting the baffle to tank wall clearance.

Impeller Type, Size and Number

The impellers for this bioreactor are identically formed to the 20000liter vessel and have a identical D/T ratio of 0.44. The bottom impelleris a Lightnin's A315 at 710 mm of diameter and the top impeller is aLightnin's A310 at 710 mm of diameter.

The Impeller Spacing, D_(c), D_(s) and D_(o)

The impeller spacing, D_(s), between the centre-line of the top impellerand the centre-line of the lower impeller is 1.229×D_(bottom) or 872 mm.The off bottom impeller clearance, D_(c) is 0.75×D_(bottom) or 531 mm.This allows the lower impeller to remain submerged at the lowestpost-inoculation volume of 2153 liters and both impellers submerged at3367 liters with liquid head above the upper impeller (D_(o)) of0.5×D_(top) or 358 mm.

Table 12 highlights the volumes that will form liquid surfaces or lowerliquid cover above the impeller. Agitation can be modified to avoidfoaming at these critical volumes.

TABLE 12 Key operating volumes that cause interaction with impellers andliquid surface Interaction Volume, L Potential Operation Submerge topimpeller 3433 Volume seen during inoculation of 1 in 5 processes with0.5D_(A310) liquid cover Liquid surface touching 2758 Volumes seenduring pre-inoculation fill of 1 in 5 top edge of top impellerprocesses. Liquid surface touching 2621 Volumes seen duringpre-inoculation fill of 1 in 5 bottom edge of top impeller processes.Submerge bottom impeller 1654 Volumes seen during pre-inoculation fillof 1 in 5 with 0.5D_(A315) liquid processes. cover Liquid surfacetouching 1104 Volumes seen during pre-inoculation fill of 1 in 5 topedge of bottom impeller and 1 in 9 processes. Liquid surface touching650 Volumes seen during pre-inoculation fill of 1 in 5 bottom edge ofbottom and 1 in 9 processes. impeller ⁽¹⁾Minimum operating volume withlower impeller submerged is 1654 litres and minimum operating volumewith both impellers submerged is 3433 litres ⁽²⁾The operating volumerange is 1914 to 3846 litres.

The 4000 l bioreactor can operate at two discrete post-inoculationvolumes with either the lower impeller submerged (during cultivation of1 in 9 seeding process) or with both impellers submerged (during 1 in 5seeding process), table 13 shows the liquid cover obtained for the upperand lower impeller during its operation.

A liquid cover of 0.67 to 0.82×D_(bottom) above the lower A315 impelleris observed during cultivation of the 1 in 9 seeded processes. This iswithin the recommendations of 0.5 to 1×D.

A liquid cover of 0.06 to 0.78×D_(top) above the top A310 impeller isobserved during cultivation of 1 in 5 seeded process. The lower liquidcover is outside the recommendation. However this liquid cover isobserved during pre-inoculation when mixing and agitation are lesscritical.

TABLE 13 Key operating volumes and the liquid cover above top impeller,Do and bottom impeller, D_(Bo) Cylinderical Do, D_(Bo), Do as ratioD_(Bo) as ratio Operating volume, L height, H (mm) mm mm of D_(A310) ofD_(A315) Post-Inoculation and  863 or 34″ — 614 or — 0.82D_(A315)Pre-Transfer, 2153 L 24″ Post-Inoculation and 1501 or 59″ 380 —0.53D_(A310) — Pre-Transfer, 3478 L or 15″ Post-Inoculation and 1678 or66″ 557 — 0.78D_(A310) — Pre-Transfer, 3846 L or 22″ Pre-Inoculation,1914 L  748 or 29″ — 499 or — 0.67D_(A315) 20″ Pre-Inoculation, 2782 L1166 or 46″ 45 or — 0.06D_(A310) — 2″ Pre-Inoculation, 3077 L 1308 or52″ 187 — 0.26D_(A310) — or 7″ ⁽¹⁾Off bottom impeller clearance, Dc =531 mm (0.75D_(A315)), Impeller separation, Ds = 872 mm (1.229D_(A315)),tank ID of 1626 mm and Height of ASME F&D base plate, H_(h) = 282 mm⁽²⁾Do = H − Ds − (Dc − H_(h)) and D_(Bo) = H − (Dc − H_(h))Agitation Rate—Rpm, P/V and Tip Speed

Table 14 specifies the agitation rate for the 4000 liter bioreactor. Thebioreactor will be agitated typically at 20-260 W/m⁻³, preferably at55-85 W/m⁻³. The agitation strategy was developed during the 500 literpilot fermentations. An agitation rate of 0 to 88±1 rpm is thereforeused as an operational range.

TABLE 14 Agitation rate for the 4000 L bioreactor Agitation rate, rpmPower per unit volume, W/m3 Tip Speed, m/s ¹0-88 0-150 0.0-3.3 ²0-860-150 0.0-3.2 ¹When both impellers submerged ²When bottom impellersubmergedMechanical Seals Specification

A double mechanical seal that is condensate lubricated is used asdescribed.

Bioreactor Aeration Requirement

Table 15 shows the gas flow, based upon scale up of constant superficialgas velocity, for DOT and pH control during the inoculum expansion inthe 4000 liter bioreactor. Oxygen is not required for DOT control.However oxygen enriched air can be used to facilitate lower gassing toprevent excess foaming. It is recommended that a smaller range N₂ MFCshould supply nitrogen for early DOT control and reducing deviant, highlevels of DOT.

TABLE 15 Gas flow rate and MFC operating ranges for the 4000 litrebioreactor Gas Operating range Comments Head Space¹ 1. Clean air 1.0-200 SLPM 1. Head space purging of CO₂ and O₂ 2. Nitrogen 2. Utilityrated 2. For rapid DOT probe zeroing 3. Helium 3. Utility rated 3. Tankintegrity testing Control Sparger 1. Clean air¹ 1. 10-60 SLPM 1. Gasflow under DOT control 2. Oxygen 2. 1.0-10 SLPM 2. Gas flow under DOTcontrol 3. Carbon dioxide 3. 1.0-20 SLPM 3. Gas flow under pH control 4.Nitrogen² 4. 2.0-15 SLPM 4. Early DOT control by ballast 5. Helium 5.Utility rated 5. Tank integrity testing ¹The air and nitrogen gas flowinto bioreactor via a bypass for post SIP tank pressurisation. ²Nitrogendelivered via the 2 to 15 SLPM N₂ MFC and could be used during early DOTcontrol

The calculation of hole size and number of holes, for the flutedsparger, is iterated until the target Reynolds number of gas emergingfrom holes is <2000 and the Sauter mean bubble diameter for a bubblechain regime is approximately 10 mm.

Table 16 show the key sparger design specification for the 4000 literbioreactor. The sparger length, S_(L) of 568 mm is determined for pipegeometry. The holes are distributed on either end of the sparger toprevent bubble liberating directly under the A315 hub. Alternatively acrescent geometry can be used. The pipe diameter is selected to aidspacing of the desired number of holes. The diameter is 38 mm. The 100 2mm holes are located on the dorsal surface of the sparger with a single2 mm hole located on the ventral surface to aid free CIP drainage of thesparger.

The bioreactor port for sparger installation is designed to a fit pipedesign of diameter of 38 mm. The position of the port allows theplacement of a control sparger at a distance of 194 mm, D_(c)−S_(c)below the bottom edge of the lower impeller and no greater 337 mm fromtank bottom, S_(c).

TABLE 16 Design specification for the 4000 litre bioreactor spargerParameter Control Sparger Gas flow, SLPM 105 Number of sparge holes 100Orifice diameter, d_(o), m 0.002 Gas flow, m³ · s⁻¹ 1.75E−03 Orificearea, m² 3.14E−06 Total orifice area, m² 3.14E−04 Density of air, Kg ·m⁻³ 1.166 Viscosity, Nm · s⁻² 1.85E−05 Sauter mean diameter, mm (d_(vs)= 1.17 V_(o) ^(0.4) d_(o) ^(0.8) g^(−0.2)) 10.21 Gravitionalacceleration, g, m · s⁻² 9.807 Density difference, Kg · m⁻³ 1048.834Reynold's number, >2000 jetting regime 704 Gas velocity at sparger,V_(o), m/s 5.57 Sparger length, S_(L), m 0.568 Combined length to drillrequired holes, m 0.2 Number of rows to fit required holes in lengthS_(L) 1 Sparger to tank bottom clearance, Sc, m 0.337 (13″) Sparger tobottom impeller clearance, Dc-Sc, m 0.194 (8″) Position of Probes, Addition and Sample Ports

The design basis for positioning of probes, addition and sample portshas been covered in example 1 and are listed in table 17:

TABLE 17 Probe, addition and sampling port specification for the 4000litre bioreactor ²Diameter, ¹Position, Probe/Port Location mm (inches)mm (inches) Rational Temperature (main) Lower ring 38.1 (1.5″) 1.) 531(21″) In the plane of 2.) 30° centre-line of bottom impeller TemperatureLower ring 38.1 (1.5″) 1.) 531 (21″) In the plane of (backup) 2.) 170°centre-line of bottom impeller pH (main) Lower ring 38.1 (1.5″) 1.) 531(21″) In the plane of 2.) 10° centre-line of bottom impeller pH (backup)Lower ring 38.1 (1.5″) 1.) 531 (21″) In the plane of 2.) 20° centre-lineof bottom impeller DOT (main) Lower ring  25.0 (0.98″) 1.) 531 (21″) Inthe plane of 2.) 150° centre-line of bottom impeller DOT (backup) Lowerring  25.0 (0.98″) 1.) 531 (21″) In the plane of 2.) 160° centre-line ofbottom impeller Spare-1 (nutrient) Lower ring  25.0 (0.98″) 1.) 531(21″) In the plane of 2.) 170° centre-line of bottom impeller Spare-2(pCO₂) Lower ring 38.1 (1.5″) 1.) 531 (21″) In the plane of 2.) 180°centre-line of bottom impeller Spare-3 (biomass) Lower ring 50.8 (2″)  1.) 531 (21″) In the plane of 2.) 190° centre-line of bottom impellerSample valve Lower ring 12.7 (0.5″) 1.) 531 (21″) NovAseptic type (main)2.) 40° Alkali addition Lower ring 50.8 (2″)   1.) 531 (21″)Diametrically 2.) 190° opposite pH probes Feed 1 Lower ring 50.8 (2″)  1.) 531 (21″) Diametrically 2.) 200° opposite pH probes Feed 2 Lowerring 50.8 (2″)   1.) 531 (21″) Diametrically 2.) 210° opposite pH probesAntifoam addition Head plate 50.8 (2″)   1.) N/A Liquid surface/ 2.)170° 0.25T from tank centre Spare surface addition Head plate 50.8(2″)   3.) N/A Liquid surface 4.) 180° directed to vessel wall DOTcontrol sparger N/A 50.8 (2″)   337 (13″) orifice 1.) 0° Overlay gasHead plate 101.6 (4″)   1.) N/A Diametrically 2.) 135° opposite vent outExhaust vent out Head plate 50.8 (2″)   1.) N/A Diametrically 2.) 315°opposite overlay gas in Transfer valve Base plate 76.2 (3.0″) 1.) N/ANovAseptic type 2.) Centre to allow free draining Inoculum transfer Headplate 101.6 (4″)   1.) N/A Directed into from 1000 L to 4000 L 2.) 320°vessel wall Media inlet Head plate 101.6 (4″)   3.) N/A Directed into4.) 310° vessel wall CIP - Spray ball Impeller 76.2 (3″)   1.) N/ACIP'ing of highest flange plate 2.) 270° point CIP - Spray ball Headplate 76.2 (3″)   1.) N/A 2.) 60° CIP - Spray ball Head plate 76.2(3″)   1.) N/A 2.) 180° CIP - Spray ball Head plate 76.2 (3″)   1.) N/A2.) 300° Pressure indicating Head plate 38.1 (1.5″) 1.) N/A transmitter(PIT) 2.) 60° Pressure gauge Head plate 38.1 (1.5″) 1.) N/A 2.) 50°Rupture disc Head plate 101.6 (4″)   1.) N/A 2.) 280° Spare nozzle Headplate 101.6 (4″)   1.) N/A 2.) 160° Sight glass Head plate 101.6 (4″)  1.) N/A 2.) 70° Light glass Head plate 76.2 (3″)   1.) N/A 2.) 75°Agitator head/flange Head plate 813 (32″) N/A Entry/removal Impellershaft and Agitator 152 (6″)  N/A Entry/removal seal manway head/ flange¹Measured from the tangential line of the base plate. Degrees pertain toplane of clockwise rotation. ²Diameter of nozzle at bioreactor.Addition Ports, Surface and Sub-Surface

The need to categorise additions ports that terminate at liquid surfaceand those that are subsurface is determined by the operational scenariosand effects of feed strategy on process control.

The 4000 liter bioreactor has been designed to accept two subsurfacefeeds and alkali that need to be discharged in well-mixed area of thebioreactor. The foam is controlled by surface addition of 1 in 10diluted C-emulsion. A single spare above surface addition port directedto the vessel wall is also designed for future flexibility. Thesplashing of culture onto the surface of the medium during inoculationof the seed bioreactor can be avoided to prevent build up of foam.Therefore the inoculum addition port is above surface and directed tothe vessel wall. The use of the harvest port in the base plate is theideal port for removal of inoculum during transfer of inoculum.Additionally the medium addition port is directed to the vessel wall. Insummary the total addition ports are:

-   -   Four surface additions with medium inlet, inoculum inlet and a        spare small addition directed to the vessel wall and addition        port for antifoam dropped on to the liquid surface away from the        vessel wall.    -   Three subsurface additions for feeds and alkali.        Sample Ports

The sample port design is similar to that specified for the 20 000 literbioreactor.

Volume Measurement

The level sensor is able to measure up to 4000 liters with an accuracy±0.5% of full span.

Bioreactor Temperature Control

The 1914 to 3077 liter of medium are brought to operating temperature,typically 36.5° C. by process control. This is achieved by “gentle”heating of the jacket and avoid high temperature at vessel wall.

Jacket

The bioreactor jacket area is specified with the followingconsiderations in mind:

-   -   Steam sterilisation at 121-125° C.    -   Warming up of 1914-3077 liters of medium from 10° C. to 36.5° C.        in <2 h.    -   All points within the bioreactor must reach ±0.2° C. of set        point, typically 36.5° C. as measured by thermocouples.    -   Chilling of 1914-3077 liters of medium from 36±2° C. to 10° C.        in 2 h.        Bioreactor pH Control

The process pH is monitored and controlled with probes connected via atransmitter to a DCS based process controller. The process pH iscontrolled by addition of CO₂ through the control sparger to bring thepH down to set point and addition of alkali to bring pH up to set point.

Alkali is added through at least one subsurface port at centre-line ofthe bottom impeller. The CO₂ will be added via the control sparger.

Control and backup probes are in the lower port ring at 531 mm from tankbottom as shown in table 17.

Bioreactor DOT Control

Dissolved oxygen is monitored and controlled with polarographic DOTprobe. The DOT set point maintained by sparging:

-   -   Initial N₂ ballast and/or air on demand.    -   Air ballast with air on demand.    -   Air ballast with oxygen on demand.

DOT control allows DOT set point to be maintained throughinterchangeable use of oxygen or air as demand gas. It is not envisagedthat pCO₂ control is required in the inoculum bioreactor. Control andbackup probes are in the lower port ring at 531 mm from tank bottom asshown in table 17.

Feed Addition Control

The point of addition is 531 mm from tank bottom, in the plane of thecentre-line of the lower impeller to assist in the rapid dissipation offeed bolus.

Antifoam Addition Control

The addition point is at surface projecting 0.25×T toward the tankcentre or 407 mm from centre of tank.

Example 3 1000 Liter Bioreactor Specification

Vessel Geometry

The vessel geometry for the 1000 liter bioreactor was determined by aniterative design basis in which the maximum working volume, freeboardstraight side distance, aspect ratio (H_(L)/T) and impeller to tankdiameter ratio (D/T) are altered until an acceptable aspect ratio isachieved.

Bioreactor Aspect Ratio H_(L)/T

Table 18 below describes the aspect ratios in the 1000 liter bioreactorat various operating volumes during normal processing. These aspectratios arise from the selection of tank ID and the operating volumerequired. From a processing perspective the mixing requirements at thedifferent operating conditions are different. During pre-inoculationstage the bioreactor mixing is important to allow medium to equilibratewith minimal K_(L)a requirement. However with post-inoculation andpre-transfer stages both mixing and K_(L)a are important considerations.Therefore both of these features were tested at the aspect ratio range.

TABLE 18 Key operating volumes and aspect ratios in the 1000 litrebioreactor Liquid Volume, L head, mm Aspect ratio, H_(L)/T Stage N-3 250484 0.56 Pre-Inoculation Stage N-3 300 570 0.66 Post Inoculation &Pre-transfer/Harvest Stage N-2 1.) 400¹ 1.) 740 1.) 0.86Pre-Inoculation, Post- 2.) 50-100¹ 2.) 143-228 2.) 0.17-0.26 drainPre-refill 3.) 192² 3.) 385 3.) 0.45 Stage N-2 1.) 450 1.) 826 1.) 0.96Post Inoculation & 2.) 450-900³ 2.) 826-1594 2.) 0.96-1.84Pre-transfer/Harvest 3.) 960⁴ 3.) 1696 3.) 1.96 ¹Pre-inoculation volumeand rolling seed inoculation volume for the 1 in 9 sub-cultivationprocess. ²Rolling seed inoculation volume for the 1 in 5 sub-cultivationprocesses. ³Rolling seed post inoculation & pre-transfer volume for the1 in 9 sub-cultivation processes. ⁴Rolling seed post inoculation &pre-transfer volume for the 1 in 5 sub-cultivation processes.Tank diameter

The tank diameter is altered to obtain the optimal aspect ratio H_(L)/T.Changes to tank internal diameter are limited by acceptable aspect ratioand plant footprint. The tank ID is 0.864 m.

Tank Height

The tank height is determined from the maximum operating volume, aspectratio H_(L)/T, freeboard straight side length, base and top platedesign. The final tank height is a compromise value determined fromvolumetric contingency for foam, plant height and acceptable impellershaft length. The head to base tangent line height is 2.347 m.

Freeboard Height

The freeboard height of 500 mm (293 liters or 31% v/v of the maximumoperating volume) is used for this seed bioreactor.

Head and Base Plate

The base and head plate design is ASME F&D for this seed bioreactor.

Bioreactor Agitation Requirement

Agitation of the bioreactor is to achieve rapid mixing, maintainhomogeneity, maintain mammalian cells in suspension and gas bubbledispersion. The underlying issue with achieving the above objectives isto minimise cell damage through shear forces originating from impellergeometry and “eddies” or vortices created behind the impeller blades. Acompromise of the above objectives was achieved by selection of anappropriate impeller type and gassing strategy.

Bottom Versus Top Driven Shaft

The motor drive is top mounted for the benefits as already highlighted.

Baffles

The baffle requirement for a centre mounted impeller is critical toprevent vortex formation. The critical issues related to baffles arebaffle number, baffle width (W), baffle length (H_(baffle)) and baffleto tank wall clearance (W_(c)).

Four equally spaced baffles that are 0.1×T or 86 mm wide 1.1×H−H_(h) or2099 mm tall and have a baffle to tank wall clearance, W_(c) of 0.01×Tor 9 mm were used.

The thickness of baffle is not specified but the thickness needs toensure rigidity to the radial component of the fluid flow. Additionallythickness needs to ensure the baffle plates are not warped during SIPthereby affecting the baffle to tank wall clearance.

Impeller Type, Size and Number

The impellers for the 1000 l bioreactor should be identical formed tothe 20 000 liter vessel with an identical D/T ratio. Therefore thebottom impeller is a Lightnin's A315 at 381 mm diameter and the topimpeller is a Lightnin's A310 at 381 mm diameter.

The Impeller Spacing, D_(c), D_(s) and D_(o)

The impeller spacing (D_(s)) between the centre-line of the top impellerand the centre-line of the bottom impeller is 2×D_(bottom) (762 mm). Theoff bottom impeller clearance (D_(c)) is 0.4×D_(bottom) (152 mm). Thisallows the bottom impeller to remain submerged with liquid cover (D_(o))of 0.5×D_(bottom) or 190 mm at the lowest post-inoculation volume of 167liters and both impeller submerged at 616 liters with liquid head abovethe upper impeller, D_(o), of 0.5×D_(top) (190 mm).

Table 19 highlights volumes that will form liquid surfaces or lowerliquid cover above the impeller Agitation can be modified to avoidfoaming at these critical volumes.

TABLE 19 Key operating volumes that cause interaction with impellers andliquid surface Interaction Volume, L Potential Operation Submerge topimpeller with 616 Volume seen during inoculation of 1 in 5 processes0.5D_(A310) liquid cover and rolling operation of the 1 in 9 processLiquid surface touching top 512 Volume seen during inoculation of 1 in 5processes edge of top impeller and rolling operation of the 1 in 9process Liquid surface touching bottom 492 Volume seen duringinoculation of 1 in 5 processes edge of top impeller and rollingoperation of the 1 in 9 process Submerge bottom impeller 167 Volume seenduring inoculation of 1 in 5 processes with 0.5D_(A315) liquid cover androlling operation of the 1 in 9 process Liquid surface touching top 90Volume seen during rolling operation of the 1 in edge of lower impeller9 process Liquid surface touching bottom 21 Volumes seen duringpre-inoculation fill of 1 in 5 edge of lower impeller and 1 in 9processes.

The 1000 l bioreactor operates at two discrete post-inoculation volumeswith either the bottom impeller submerged during the 1 in 5 processesand 1 in 9 processes or with both impellers submerged during the N-2phase of the 1 in 5 process and rolling seed operations for both 1 in 5and 1 in 9 processes.

Table 20 shows the liquid cover above the upper and lower impellerduring operation of the 1 in 5 and 1 in 9 sub-cultivation processes.During rolling operation of the 1 in 5 and 1 in 9 processes the liquidcover above the lower impeller falls below 0.5×D. It is thereforeimportant to reduce the agitation rate, to avoid surface gasentrainment, whilst operating at this low volume. At 960 liters a liquidcover, (D_(o)) of 2.05×Dtop is obtained. At this level K_(L)a has beenshown not to be adversely affected and bulk blending is not an issue.

TABLE 20 Key operating volumes and the liquid cover above top impeller,Do and bottom impeller, D_(Bo) Cylinderical Do, D_(Bo), Do as ratioD_(Bo) as ratio Operating volume, L height, H (mm) mm mm of D_(A310) ofD_(A315) Pre-Inoculation, 250 L 334 — 332 — 0.87D_(A315)Pre-Inoculation, 400 L 590 — 588 — 1.54D_(A315) Post-Inoculation and 419— 417 — 1.10D_(A315) Pre-Transfer, 300 L Post-Inoculation and 675 — 673— 1.77D_(A315) Pre-Transfer, 450 L Post drain, pre-bulk 235 — 233 —0.61D_(A315) 192 L Post drain, pre-bulk 50-100 L 0-78 —  76 —0.2D_(A315) Post-Inoculation and 1443  679 — 1.78D_(A310) —Pre-Transfer, 900 L Post-Inoculation and 1545  782 — 2.05D_(A310) —Pre-Transfer, 960 L ¹Off bottom impeller clearance, Dc = 152 mm(0.4D_(A315)), Impeller separation, Ds = 762 mm (2D_(A315)), tank ID of864 mm and Height of ASME F&D base plate, H_(h) = 151 mm ²Do = H − Ds −(Dc − H_(h)) and D_(Bo) = H − (Dc − H_(h))Agitation Rate—Rpm, P/V and Tip Speed

Table 21 specifies the agitation rate for the 1000 liter bioreactor. Thebioreactor is agitated at around 20-260 W/m³, preferably at 55-85 W/m³.The agitation strategy was developed during the 500 liter pilotfermentations. An agitation rate of up to 155±1 rpm is used as anoperational range.

TABLE 21 Agitation rate for the 1000 L bioreactor Tip Agitation rate,rpm Power per unit volume, W · m⁻³ Speed, m · s⁻¹ ¹0-155 0-150 3.1²0-145 0-145 2.9 ¹When both impellers submerged ²When bottom impellersubmergedMechanical Seals Specification

A double mechanical seal that is condensate lubricated as described wasused.

Bioreactor Aeration Requirement

Table 22 shows the gas flows based upon scale up of constant superficialgas velocity, for DOT and pH control during the inoculum expansion inthe 1000 liter bioreactor. Oxygen will not be required for DOT control.However oxygen enriched air may be used to facilitate lower gassing toprevent excess foaming. It is recommended that the smaller range CA MFCshould be used to delivery nitrogen for early DOT control and reducingdeviant, high levels of DOT.

TABLE 22 Gas flow rate and MFC operating ranges for the 1000 litrebioreactor Gas Operating range Comments Head Space¹ 1. Clean air 1. 0-50SLPM 1. Head space purging of CO₂ and O₂ 2. Nitrogen 2. Utility rated 2.For rapid DOT probe zeroing 3. Helium 3. Utility rated 3. Tank integritytesting Control Sparger 1. Clean air¹ 1. 2-20 SLPM 1. Gas flow under DOTcontrol 2. Oxygen 2. 0.2-5 SLPM 2. Gas flow under DOT control 3. Carbondioxide 3. 0.2-10 SLPM 3. Gas flow under pH control 4. Nitrogen² 4.0.2-5 SLPM 4. Early DOT control by ballast 5. Helium 5. Utility rated 5.Tank integrity testing ¹The air and nitrogen gas flow into bioreactorvia a bypass for post SIP tank pressurisation. ²Nitrogen delivered viathe 0 to 5 SLPM CA MFC, could be used during early DOT control.

The calculation of hole size and number is iterated until the targetReynolds number of gas emerging from holes is <2000 and the Sauter termean bubble diameter for a bubble chain regime is approximately 10 mm.

Table 23 shows the key sparger design specification for the 1000 literbioreactor. The sparger length, S_(L) of 305 mm is determined for pipegeometry. The holes are distributed on either end of the sparger toprevent bubble liberating directly under the A315 hub. Alternatively acrescent geometry can be considered.

The pipe diameter is 25 mm. 30 2 mm holes are located on the dorsalsurface of the sparger with a single 2 mm hole located on the ventralsurface to aid free CIP drainage of the sparger.

The bioreactor port for sparger installation is designed to fit pipedesign of diameter of 25 mm. The position of port allows the placementof control sparger at a distance of 88 mm (D_(c)−S_(e)) below the bottomedge of the bottom impeller and no greater than 64 mm from tank bottom(S_(c)).

TABLE 23 Design specification for 1000 litre bioreactor spargersParameter Control Sparger Gas flow, SLPM 35 Number of sparge holes 30Orifice diameter, d_(o), m 0.002 Gas flow, m³ · s⁻¹ 5.83E−04 Orificearea, m² 3.14E−06 Total orifice area, m² 9.42E−05 Density of air, Kg ·m⁻³ 1.166 Viscosity, Nm · s⁻² 1.85E−05 Sauter mean diameter, mm (d_(vs)= 1.17 V_(o) ^(0.4) d_(o) ^(0.8) g^(−0.2)) 10.65 Gravitionalacceleration, g, g m · s⁻² 9.807 Density difference, Kg · m⁻³ 1048.834Reynold's number, >2000 jetting regime 782 Gas velocity at sparger,V_(o), m/s 6.19 Sparger length, S_(L), m 0.305 Combined length to drillrequired holes, m 0.06 Number of rows to fit required holes in lengthS_(L), m 1 Sparger to tank bottom clearance, Sc, m 0.064 Sparger tobottom impeller clearance, Dc-Sc, m 0.088Position of Probes, Addition and Sample Ports

The design basis for positioning of probes, addition and sample ports isthe same as for the 20 000 l bioreactor.

TABLE 24 Probe, addition and sampling port specification for the 1000litre bioreactor Diameter, mm ¹Position, mm Probe/Port Location (inches)(inches) Rational Temperature Lower 38.1 (1.5″) 1. 286 (11″) Positionedto minimise (main) ring 2. 30° monitored volume Temperature Lower 38.1(1.5″) 1. 286 (11″) Positioned to minimise (backup) ring 2. 170°monitored volume PH (main) Lower 38.1 (1.5″) 1. 286 (11″) Positioned tominimise ring 2. 10° monitored volume PH (backup) Lower 38.1 (1.5″) 1.286 (11″) Positioned to minimise ring 2. 20° monitored volume DOT (main)Lower  25.0 (0.98″) 1. 286 (11″) Positioned to minimise ring 2. 150°monitored volume DOT (backup) Lower  25.0 (0.98″) 1. 286 (11″)Positioned to minimise ring 2. 160° monitored volume Spare-2 (spare -Lower 38.1 (1.5″) 1. 286 (11″) Positioned to minimise pCO₂) ring 2. 180°monitored volume Spare-3 (spare - Lower 50.8 (2″)   1. 286 (11″)Positioned to minimise Biomass) ring 2. 190° monitored volume Samplevalve Lower 38.1 (1.5″) 1. 286 (11″) NovAseptic type (main) ring 2. 40°Sample valve Lower 38.1 (1.5″) 1. 286 (11″) NovAseptic type (back up)ring 2. 40° Alkali addition Lower 12.7 (0.5″) 1. 286 (11″) Diametricallyopposite ring 2. 190° pH probes Feed 1 Lower 12.7 (0.5″) 1. 286 (11″) Diametrically opposite ring 2. 200° pH probes Feed 2 Lower 12.7(0.5″) 1. 286 (11″)  Diametrically opposite ring 2. 210° pH probesAntifoam addition Head 50.8 (2″)   1. N/A Liquid surface/0.25T plate 2.170° from tank centre Spare surface Head 50.8 (2″)   1. N/A Liquidsurface directed addition plate 2. 180° to vessel wall DOT controlsparger N/A 50.8 (2″)   1. 64 (2.5″) orifice 2. 0° Overlay gas Head 38.1(1.5″) 1. N/A Diametrically opposite plate 2. 135° vent out Exhaust ventout Head 38.1 (1.5″) 1. N/A Diametrically opposite plate 2. 315° overlaygas in Transfer valve Base 50.8 (2.0″) 1. N/A NovAseptic type to plate2. Centre allow free draining Media inlet Head 76.2 (3″)   1. N/ADirected into vessel plate 2. 310° wall Inoculum transfer Head 50.8(2.0″) 1. N/A Directed into vessel from S200 to plate 2. 320° wall 1000L CIP - Spray ball Head 76.2 (3″)   1. N/A CIP'ing of highest plate 2.270° point CIP - Spray ball Head 76.2 (3″)   1. N/A plate 2. 60°Pressure gauge Head 38.1 (1.5″) 1.) N/A plate 2.) 50° Rupture disc Head50.8 (2″)   1.) N/A plate 2.) 280° Spare nozzle Head 101.6 (4″)   1.)N/A plate 2.) 160° 1. Hand hole Head 1. 203.2 (8″)   1.) N/A Single portpermitting 2. Sight glass plate 2. 101.6 (4″)   2.) 70° two functionsAgitator shaft Head 152.4 (6″)  1.) N/A Centre of head plate openingplate 2.) 75° ¹Measured from the tangential line of the base plate.Degree pertains to plane of clockwise rotation. ²Diameter of nozzle atthe bioreactor

In order to monitor, control and sample from a volume of 50 l, theprobes and port ring needs to be 151 mm from tank bottom. However theprobe/port ring cannot be located this low as it falls on the weld ofthe base plate and the straight cylindrical side of the bioreactor. Theprobe and port ring has been specified at 286 mm from tank bottom. Thispermits a volume of 134 liters to be monitored, controlled and sampled.The probes/port ring is located as close to the tank bottom as permittedto minimise the monitored/controlled volume.

Addition Ports, Surface and Sub-Surface

The 1000 liter bioreactor has been designed to accept two subsurfacefeeds and alkali to be discharged into a well-mixed area of thebioreactor. The foam is controlled by surface addition of 1 in 10diluted C-emulsion. A single above surface spare addition port directedto the vessel wall was also integrated for future flexibility. Thesplashing of culture on to the surface of the medium during inoculationof seed bioreactor should be avoided to prevent build up of foam.Therefore the inoculum addition port is above surface and directed tothe vessel wall. The use of the harvest port in the base plate is theideal port for removal of inoculum during transfer of inoculum.Additionally the medium addition port is directed on to the vessel wall.In summary the total addition ports are:

-   -   Four surface additions with medium inlet, inoculum inlet and a        spare small addition directed to the vessel wall and addition        port for antifoam dropped on to the liquid surface away from the        vessel wall.    -   Three subsurface additions for feeds and alkali.        Sample Ports

The sample port design is similar to that specified for the 20 000 literbioreactor. The sample port is located 286 mm from tank bottom tominimise the volume that can be sampled.

Volume Measurement

The level sensor is able to measure up to 1000 liters. The level sensorsensitivity is at least 0.25% of full span.

Bioreactor Temperature Control

The 250 to 800 liters of medium is brought to operating temperature,typically 36.5° C. during initial inoculation and “seed rollingoperation” by process control. This is achieved by “gentle” heating ofthe jacket and avoid high temperature at vessel wall.

Jacket

The bioreactor jacket area is specified with the followingconsiderations in mind:

-   -   Steam sterilisation at 121-125° C.    -   Warming up of 250-800 liters of medium from 10° C. to 36.5° C.        in <2 hrs.    -   All points within the bioreactor must reach ±0.2° C. of set        point, typically 36.5° C. as measured by thermocouples.    -   Chilling of 400 liters of medium from 36±2° C. to 10° C. in 2        hrs.        Bioreactor pH Control

The process pH is monitored and controlled with probes connected via atransmitter to a DCS based process controller. The process pH iscontrolled by addition of CO₂ to bring the pH down to set point andaddition of alkali to bring pH up to set point. Alkali is added throughat least one subsurface port at centre-line of the bottom impeller. TheCO₂ is added via the control sparger.

The control and back up probes are in the lower port ring at 286 mm fromtank bottom to minimise the volume that can be monitored as shown inTable 24.

Bioreactor DOT Control

Dissolved oxygen is monitored and controlled with polarographic DOTprobe. The DOT set point maintained by sparging:—

-   -   Initial N₂ ballast and/or air on demand    -   Air ballast with air on demand    -   Air ballast with oxygen on demand

DOT control allows DOT set point to be maintained throughinterchangeable use of oxygen or air as demand gas.

Control and back up probes are in the lower port ring at 286 mm fromtank bottom minimise the volume that can be monitored, as shown in table24.

Feed Addition Control

The point of addition is 286 mm from tank bottom, in the vicinity of thecentre-line of the bottom impeller to assist in the rapid dissipation offeed bolus.

Antifoam Addition Control

The addition point is at surface projecting 0.25×T toward the tankcentre or 216 mm from tank centre.

Example 4 Bioreactor Train

The bioreactor design is based on the ability to perform both 1 in 5(20% v/v) and 1 in 9 (11% v/v) subculture ratios. The bioreactor trainconsists of a 1000 liter (Stages N-3 and N-2) and 4000 liter (Stage N-1)seed bioreactors followed by a 20 000 liter production bioreactor (StageN). The operating volumes for each bioreactor are defined in examples 1to 3. The bioreactors are based on a stirred tank design and a topdriven agitator system was used.

The design is based on the need to ensure a homogenous environment withrespect to process parameters such as pH, dissolved oxygen tension (DOT)and temperature, maintaining a well mixed cell suspension and blendingnutrient feeds within the bioreactor. This provides the necessaryphysicochemical environment for optimal cell growth, productaccumulation and product quality. Key to the design philosophy is theneed to maintain geometric similarity. This allows a scale down model tobe developed at 12 liter laboratory and 500 liter pilot scales. Thedesign of the seed and production bioreactors are based on the sameprinciples although some departures are required to allow forflexibility in processing. The aspect ratios (H_(L)/T) selected aretypical of those used in mammalian cell culture and are in the range0.17 to 1.96 post-inoculation.

TABLE 25 Key bioreactor design parameters 1000 litre 4000 litre 20000litre Aspect ratio (H_(L)/T) 0.17-1.96 0.63-1.21 0.83-1.34 Impeller totank 0.44-0.46 0.44-0.46 0.44-0.46 diameter (D/T) Operating Volume 50-960 1914-3846 13913-21739 (L) Agitator speed  0-155  0-88  0-80(rpm) Control sparger  2-20  0-60   0-1000 CA (SLPM) Ballast sparger Noballast sparger No ballast sparger  0-500 CA/N₂ flow (SLPM) Cultivation2-5¹ 2-5 10-15 residence time (days) Feed additions 2 surface 2 surface4 surface 3 sub-surface 3 sub-surface 4 sub-surface ¹The cultureresidence time in 1000 litre bioreactor may be higher depending on thelength of time the bioreactor is repeatedly sub-cultivated or “rolled”.

The design constraint is based upon a seeding ratio of 11% v/v (1 in 9dilution) and 20% v/v (1 in 5 dilution), with feed application of 4% v/vto 25% v/v of the post-inoculation volume. The post-inoculation volumein the production bioreactor is adjusted for feed applications up to 15%such that after the addition of all the feeds the final volume atharvest ends up at 20 000 l. However for feed applications greater then15% v/v the post-inoculation volume is adjusted for a 15% v/v feed butfollowing the application of feeds the final pre-harvest volume will bea minimum of 20 000 and a maximum 22 000 liters. The productionbioreactor is expected to hold a total of 20 000 to 22 000 liters at theend of a batch. Table 26 shows the pre-inoculation volume, inoculationvolume and transfer or harvest volume for each of the three inoculumexpansion stages and the production bioreactor.

The seed bioreactors (stage N-1 to N-3) are unlikely to be fed thereforethe maximum operating volume will be at inoculation. The operatingvolume range for the 4000 liter seed bioreactor (stage N-1) is 1914 to3846 liters. In order to design a bioreactor that can grow cells from20% v/v seed split ratio, the 1000 liter seed bioreactor (stages N-2 andN-3) will operate at two operating ranges. For the 11% v/v seed splitratio the bioreactor train can produce sufficient culture to meetforward processing cell concentration criteria in a singleexpansion/sub-cultivation stage. However the bioreactor train requirestwo expansion/sub-cultivation stages to meet forward processing criteriafor 20% v/v seed split ratio process. Thus for 11% v/v seed split ratioprocess an operating range of 400 to 450 liters is required and for the20% v/v seed split ratio process an operating volume range of 250 to 960liters is required.

TABLE 26 Vessel sizes for bioreactor train 4000 1000 litre litre 20000litre Stage N − 3 N − 2 N − 1 N 11% v/v Seed with 4 to 25% v/vproduction feed Pre-inoculation Volume (L) 400 — 1914 15456-17096Inoculation Volume (L) 450 — 2153 17391-19231 Transfer or Harvest Volume(L) 450 — 2153 20000-21739 20% v/v Seed with 4 to 25% v/v productionfeed Pre-inoculation Volume (L) 250 768 2782-3077 13913-15385Inoculation Volume (L) 300 960 3478-3846 17391-19231 Transfer or HarvestVolume (L) 300 960 3478-3846 20000-21739 Assumed operating volumeMinimum Volume (L) 250 1914 13913 Maximum Volume (L) 960 3846 21739Ratio of Maximum volume/ 3.84 2.01 1.56 Minimum volume

It is recommended that the 1000 liter seed bioreactor is inoculated fromculture produced in an S200 Wave bioreactor.

1000 l: This bioreactor is operated in batches of up to 5 days, withpotential “shot additions” of feeds, for cultivation of mammalian cells.However due to repeated drain and refill operation at the end of eachbatch the total process residence time in this bioreactor can exceed 30days. The mammalian cells are kept in a homogeneous suspension byagitation via an identical impeller system to the 20 000 literbioreactor. Additionally other features will be kept geometricallysimilar to the 20 000 liter bioreactor, where possible.

Sparging air or oxygen and air or nitrogen respectively will controlprocess DOT. Process pH is controlled by addition of alkali for basecontrol and of sparged CO₂ for acid control.

The process operating volume of the bioreactor changes at differentphases of operation. Initially the bioreactor is aseptically filled witha bolus of medium at 250 to 400 liters in 0.5 h. The bioreactor isoperated in a pre-inoculation phase to bring the process variables topredefined set points. 50 liter culture from a (N-4) S-200 seed wavebioreactor is inoculated, by pneumatic assisted flow, or pumped with aperistaltic pump in 25 to 30 minutes into the 1000 liter bioreactor at 1in 5 or 1 in 9 dilutions. The post-inoculation operating volume is 300and 450 liters for 1 in 5 and 1 in 9 seeded process respectively. Theaddition of alkali for base control and 1 in 10 antifoam suspension forsuppression of foam contributes towards the final volume. The inoculumculture may be fed by a “shot addition” if the culture interval islonger then expected. As a result of mixing and gassing the liquidvolumes described above will expand due to gas hold up. The extent ofthis rise is dependent on the sparger type used, power per unit volumeimparted by impellers and superficial gas velocity of sparged gasses.

The N-3 stage ends when viable cell concentration reaches transfercriteria. The N-2 stage for 1 in 5 process begins with a bulk up involume to 960 liter by draining of 192 liter excess culture and additionof 768 liter fresh medium in 1.5 h. 696 to 769 liter of culture aretransferred at the end of N-2 stage to the 4000 liter bioreactor for the1 in 5 processes. For the 1 in 9 processes 239 liters are transferred tothe 4000 liter bioreactor.

The 1000 l bioreactor is continuously “drained and refilled with freshmedium” or “rolled” to provide back up culture for the 4000 literbioreactor. The duration of the rolling seed operation is dependent onthe length of the production campaign and the permissible elapsedgenerations number of the seed culture. Typically it is assumed thatrolling seed operation is in excess of 30 days. The rolling operationconsists of retaining approximately 192 liters of the 960 liter cultureand diluting with 768 liter fresh medium for the 1 in 5 processes. Forthe 1 in 9 processes the 1000 liter bioreactor is expected to be“rolled” by retaining 50 to 100 liter of the 450 to 900 liter cultureand diluting with 400 to 800 liter fresh medium. Process control rangesare relaxed over this operation. The medium added to the bioreactorduring rolling operation is warmed to 30° C.

4000 l: This bioreactor is operated in batch of no more then 5 days,with potential “shot additions” of feeds, for cultivation of mammaliancells. The mammalian cells are kept in a homogeneous suspension byagitation via an identical impeller system described in example 1.Additionally this vessel is geometrically similar to the 20 000 literbioreactor.

Sparging air or oxygen and air or nitrogen respectively controls processDOT. Process pH is controlled by addition of alkali for base control andof sparged CO₂ for acid control.

The process operating volume of the bioreactor changes at differentphases of operation. Initially the bioreactor is aseptically filled witha bolus of protein free medium at 1914 to 3077 liters in 1.5 h. Thebioreactor operates in a pre-inoculation phase to bring the processvariables to predefined set points. Culture from the 1000 liter (N-2)seed seed bioreactor is inoculated by pneumatic flow at a flowrate toallow transfer in one hour, at 1 in 5 or 1 in 9 dilutions. Thepost-inoculation operating volume is 2153 to 3846 liters. The additionof alkali for base control and 1 in 10 antifoam suspension forsuppression of foam contributes towards the final volume. The inoculumculture may be fed by a “shot addition” if the culture interval islonger then expected. As a result of mixing and gassing the liquidvolume expands due to gas hold up. The extent of this rise is dependenton the sparger type used, power per unit volume imparted by impellersand superficial gas velocity of sparged gasses.

20 0001: This bioreactor is operated in batch or fed batch mode for 10to 15 days for the cultivation of mammalian cells. The mammalian cellsare kept in a homogeneous suspension by agitation via an impellersystem.

The process operating volume of the bioreactor changes at differentphases of operation. Initially the bioreactor is aseptically filled withcell culture medium at 13913 to 17096 liters in 1-2 h. The bioreactor isoperated in a pre-inoculation phase to bring the process variables topredefined set points. Culture from the 4000 liter seed bioreactor (N-1)is inoculated by pneumatic flow at a flow rate range of <4000 l/h intothe 20 000 liter bioreactor at 1 in 5 or 1 in 9 dilutions. Thepost-inoculation volume continuously increases following an applicationof sub-surface feeds to maximum of 20 000 liters (two feeds totalling 4to 25% v/v). The addition of alkali for base control and 1 in 10antifoam suspension for suppression of foam accounts for about 100liters and 20 liters respectively. As a result of mixing and gassing theliquid volume expands due to gas hold up. The extent of this rise isdepended on the sparger type used (fluted or sintered), power per unitvolume imparted by impellers and superficial gas velocity of spargedgasses.

Table 27 describes the aspect ratios in the 20 000 liter bioreactor atvarious operating volumes during normal processing. The aspect ratioshave been tested at 500 liter scale and provided the superficial gasvelocity and power per unit volume are kept constant the K_(L)a remainsconstant.

TABLE 27 Key operating volumes and aspect ratios in the 20 000 litrebioreactor Volume, L Liquid head, mm Aspect ratio, H_(L)/TPre-Inoculation 13913-17096 2458-2977 0.88-1.07 Post Inoculation17391-19231 3025-3325 1.08-1.19 Harvest 20000-21739 3451-3734 1.23-1.34

What is claimed is:
 1. A bioreactor system for the cultivation ofmammalian cells comprising: a first bioreactor having a volume ofbetween 500 and 1000 l and at least one top impeller and at least onebottom impeller, connected to a second bioreactor, wherein the at leastone top impeller is a hydrofoil impeller, wherein the first bioreactorhas an aspect ratio between 0.17 to 1.96 and an impeller to tankdiameter between 0.44 to 0.46, the second bioreactor having a volume ofbetween 2000 and 4000 l and at least one top impeller and at least onebottom impeller, connected to a third bioreactor, wherein the at leastone top impeller is a hydrofoil impeller, wherein the second bioreactorhas an aspect ratio between 0.17 to 1.96 and an impeller to tankdiameter between 0.44 to 0.46, and wherein the second bioreactor has avolume greater than the first bioreactor the third bioreactor having avolume of between 10 000 and 20 000 l and at least one top impeller andat least one bottom impeller, wherein the at least one top impeller is ahydrofoil impeller, wherein the third bioreactor has an aspect ratiobetween 0.17 to 1.96 and an impeller to tank diameter between 0.44 to0.46, and wherein the third bioreactor has a volume greater than thesecond bioreactor, wherein the first, second or third bioreactormaintain a homogeneous environment with respect to pH, dissolved oxygentension (DOT) and temperature, allows for a well-mixed cell suspensionand a blending of nutrient feeds within the bioreactor, and wherein thefirst, second and third bioreactors are geometric similar.
 2. Thebioreactor system according to claim 1, wherein the first, second orthird bioreactor is a biocompatible tank or vessel.
 3. The bioreactorsystem according to claim 1, wherein the first, second or thirdbioreactor is operated in a batch or a fed batch mode.
 4. The bioreactorsystem according to claim 1, wherein the cultivation conditions are thesame in the first, second and third bioreactors.
 5. The bioreactorsystem according to claim 1, wherein the temperature during operation isin the range from 36 to 38° C. with an accuracy of ±0.2° C.
 6. Thebioreactor system according to claim 1, wherein the at least one bottomimpeller is a hydrofoil impeller.
 7. The bioreactor system according toclaim 1, wherein the at least one top impeller is a down-flowingimpeller or an up-flowing impeller.
 8. The bioreactor system accordingto claim 7, wherein the at least one top impeller is a down-flowingaxial hydrofoil impeller.
 9. The bioreactor system according to claim 1,wherein the at least one top impeller is a three bladed hydrofoilimpeller.
 10. The bioreactor system according to claim 1, wherein the atleast one bottom impeller is a four pitched-bladed impeller.
 11. Thebioreactor system according to claim 1, wherein a top impeller powernumber (N_(p)) is at least 0.1 and at most 0.9.
 12. The bioreactorsystem according to claim 1, wherein a bottom impeller power number(N_(p)) is at least 0.5 and at most 0.9.
 13. The bioreactor systemaccording to claim 1, wherein a top impeller flow number (N_(q)) is atleast 0.4 and at most 0.9.
 14. The bioreactor system according to claim1, wherein an impeller spacing (D_(s)) between the top impeller and thebottom impeller is between 1×the diameter of the bottom impeller(D_(bottom)) and 2×D_(bottom), wherein a liquid height above the topimpeller (D_(o)) is between 0.3×the diameter of the top impeller(D_(top)) and 2.5×D_(top), and wherein a bottom clearance (D_(c))between the tank bottom and the center-line of the bottom impeller is atleast 0.35×D_(bottom).
 15. The bioreactor system according to claim 1,wherein the first, second or third bioreactor has at least one sparger.16. The bioreactor system according to claim 15, wherein the at leastone sparger fits to a pipe design.
 17. The bioreactor system accordingto claim 15, wherein the at least one sparger is a flute-type sparger oris a sintered sparger.
 18. The bioreactor system according to claim 15,wherein a clearance (S_(c)) between the at least one sparger to the tankbottom is at least 0.17×sparger length (S_(L)), and wherein a clearance(D_(c)−S_(c)) between the at least one sparger to the center-line of thebottom impeller is 0.25×sparger length (S_(L)).
 19. The bioreactorsystem according to claim 15, wherein the first, second or thirdbioreactor has a clearance (S_(c)) between the at least one sparger tothe tank bottom of between 315 mm and 360 mm, and wherein the first,second or third bioreactor has a clearance (D_(c)−S_(c)) between the atleast one sparger to the center-line of the bottom impeller of between180 mm and 205 mm.
 20. The bioreactor system according to claim 1,wherein the first, second or third bioreactor has at least one baffle.21. The bioreactor system according to claim 20, wherein the first,second or third bioreactor has a length of the at least one baffle at1.1×a total straight height of the bioreactor (H), wherein the first,second or third bioreactor has a width of the at least one baffle at0.1×the internal diameter of the tank, wherein the first, second orthird bioreactor has a baffle to tank wall clearance (W_(c)) of 0.01×theinternal diameter of the tank, and wherein the first, second or thirdbioreactor has a height of the at least one baffle (H_(baffle)) at1.1×the total straight height of the bioreactor (H)—a head height of thebioreactor (H_(h)).
 22. The bioreactor system according to claim 21,wherein said at least two ports are spatially separated from oneanother.
 23. The bioreactor system according to claim 1, wherein thefirst, second or third bioreactor has at least two ports for alkaliaddition.
 24. The bioreactor system according to claim 23, wherein oneport is located at the center-line of the bottom impeller and the otherport is located at the center-line of the top impeller.
 25. Thebioreactor system according to claim 24, wherein the first, second orthird bioreactor has at least one sparger, wherein a clearance (S_(c))between the at least one sparger to the tank bottom is between 560 mmand 620 mm, and wherein a clearance (D_(c)−S_(c)) between the at leastone sparger to the center-line of the bottom impeller is between 300 mmand 340 mm.
 26. The bioreactor system according to claim 1, wherein thesecond bioreactor has the aspect ratio between 0.63 to 1.21, and whereinthe third bioreactor has the aspect ratio between 0.83 to 1.34.